Aminoalcohol lipidoids and uses thereof

ABSTRACT

Aminoalcohol lipidoids are prepared by reacting an amine with an epoxide-terminated compound are described. Methods of preparing aminoalcohol lipidoids from commercially available starting materials are also provided. Aminoalcohol lipidoids may be prepared from racemic or stereochemically pure epoxides. Aminoalcohol lipidoids or salts forms thereof are preferably biodegradable and biocompatible and may be used in a variety of drug delivery systems. Given the amino moiety of these aminoalcohol lipidoid compounds, they are particularly suited for the delivery of polynucleotides. Complexes, micelles, liposomes or particles containing the inventive lipidoids and polynucleotide have been prepared. The inventive lipidoids may also be used in preparing microparticles for drug delivery. They are particularly useful in delivering labile agents given their ability to buffer the pH of their surroundings.

RELATED APPLICATIONS

The present application is a division of U.S. Application, U.S. Ser. No.16/208,295, filed Dec. 3, 2018, which is a continuation of U.S.Application U.S. Ser. No. 15/417,530, filed Jan. 27, 2017, which is acontinuation of U.S. Application U.S. Ser. No. 14/599,004, filed Jan.16, 2015, which is a division of U.S. Application U.S. Ser. No.13/128,020, filed Aug. 16, 2011, which is a national stage filing under35 U.S.C. 371 of International Patent Application Serial No.PCT/US2009/006018, filed Nov. 6, 2009, which claims priority under 35U.S.C. § 119(e) to U.S. provisional applications, U.S. Ser. No.61/112,414, filed Nov. 7, 2008, and U.S. Ser. No. 61/166,518, filed Apr.3, 2009; each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos. R37EB000244, R01 EB000244 and U54 CA119349 awarded by the NationalInstitutes of Health (NIH). The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Despite promise in the laboratory, the potential of genetic therapiesfor the treatment of disease has yet to be realized. Initial attempts totranslate genetic materials into cures led to cancer and, in some cases,death to patients involved in the clinical trials. Such deleteriousoutcomes were attributed not to the genetic material, but to the viraldelivery systems utilized in these trials. As a result, there has beenintense interest in developing synthetic materials that have thedelivery efficiencies of viral vectors but circumvent the mutagenesisthat led to the observed side effects (e.g., cancer).

Synthetic materials, or nonviral delivery vectors, come in a variety offorms that work in unique ways. Polymeric materials such aspolyethylenimine or poly(beta-amino ester)s have been shown toefficiently complex DNA for delivery into the cell. Polymers in theseclasses of delivery agents typically contain amine functionalities thatserve to electrostatically bind to DNA to form nanoparticles that arethen taken up by the cell via endocytosis. Once in the cell, these aminegroups serve to buffer the endosome and cause an influx of ions due tothe proton-sponge mechanism. The resulting burst of the endocyticvesicle leads to the release of the payload of the particle, which isthen free to travel to the nucleus where the DNA is expressed.

While the mechanism of RNA-based therapies is different, the objectiveof the delivery system remains the same. The RNA must be complexed andinternalized by the cell in order to exhibit activity. In many cases,polymeric materials do not work as efficiently for RNA delivery. This islikely due to the difference in chemical structure of the therapeuticRNA being delivered, which are generally short, linear fragmentscontaining additional hydroxyl moieties on each ribose ring. Thesedifferences necessitate an alternative nonviral approach that is suitedfor complexation with short RNA strands. Promising results have beenachieved with materials that form liposomes or lipoplexes that entrapthe RNA or form nanoparticles, which are efficiently internalized by thecell.

The materials utilized to form a lipid-based delivery system generallyconsist of a positively charged headgroup and a hydrophobic tail. Thecharged portion serves to electrostatically bind the negatively chargedRNA, while the hydrophobic tail leads to self-assembly into lipophilicparticles. Such cationic lipids are promising but still fall short ofthe transfection efficiency achieved by viral vectors.

Few advances have been made in the field, in part due to the limitedstructural diversity of these lipid-like molecules, which is a result ofthe difficult synthetic procedures required to access these structures.Therefore, in order to push the area of nonviral lipid particle deliverysystems forward, it is necessary to investigate chemical transformationsthat can lead to diverse molecules capable of complexing RNA andshuttling the material across the cell membrane. The most successfulapproach to date has been the contribution by Anderson and coworkers,who generated a library of lipid-like materials using straightforwardsimple chemical transformations. This set of materials was based on thewell-known and efficient reaction known as the Michael addition of anamine to an acrylamide or acrylate to yield a beta-amino amide or abeta-amino ester, respectively. These structures consist of an aminecore linked to long, hydrophobic alkyl chains. Starting with a set ofamines and Michael acceptors, the team generated over 1000 compoundsthat were tested for their ability to complex and deliver RNA in a highthroughput assay. This screen led to the identification of a number oflead compounds that were more efficient in vitro than the currentindustry standard, Lipofectamine 2000, and are currently being tested invivo for potential use in therapeutic applications (Akinc et al., Nat.Biotech. 2008, (26) 561).

There exists a continuing need for a new set of lipid-like moleculesthat feature similar properties to the existing amine-containinglipidoid materials, but accessed through an entirely different chemicalreaction and having the ability to deliver RNA as well as other nucleicacids and other diagnostic, therapeutic, and prophylactic agents tocells.

SUMMARY OF THE INVENTION

The present invention originates from the discovery that aminoalcohollipidoid compounds for drug delivery may be prepared by reacting anamine with a terminal epoxide or an aldehyde.

The inventive lipidoid compounds are particularly useful in theadministration of polynucleotides. The aminoalcohol lipidoid compoundsof the present invention are amenable to combinatorial synthesis andscreening to generate libraries of compounds for use as nonviral drugdelivery agents. The inventive compounds may be used for other purposesas well such as, for example, coatings, additives, excipients.

In one aspect, the present invention provides novel aminoalcohollipidoid compounds of the formulae:

These aminoalcohol lipidoid compounds may be prepared by reacting anamine with an epoxide-terminated compound. In certain embodiments, theepoxide is stereochemically pure (e.g., enantiomerically pure). Incertain embodiments, the amine is stereochemically pure (e.g.,enantiomerically pure). In certain embodiments, the lipidoid is preparedfrom the reductive amination of an imine which is derived from thecondensation of an amine and an aldehyde. In certain embodiments, each

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In certain embodiments, an amine and an epoxide-terminated compound arereacted at elevated temperatures in the absence of solvent to preparethe inventive aminoalcohol lipidoids as shown in FIG. 1. In certainembodiments, the aminoalcohol lipidoid compounds include a hydrophilicportion resulting from the opening of the epoxide by the amine and ahydrophobic aliphatic tail.

Typically, the amines chosen contain between two and five amine moietiesand the epoxide-terminated compounds include a tail of varying chainlengths and optionally feature various functional groups and varyingdegrees of saturation. The inventive aminoalcohol lipidoid compounds maybe used in the delivery of therapeutic agents (e.g., polynucleotide,small molecule, protein, peptide) to a subject. The inventiveaminoalcohol lipidoid compounds are particularly useful in deliveringnegatively charged agents given the tertiary amines available forprotonation thus forming a cationic moiety. For example, theaminoalcohol lipidoid compounds may be used to delivery DNA, RNA, orother polynucleotides to a subject or to a cell. As would be appreciatedby one of skill in the art, the above reaction may result in a mixturewith lipidoid compounds having one tail, some having two tails, somehaving three tails, and yet others having four or more tails. Also, twodifferent epoxide compounds may be used in the reaction mixture toprepare an aminoalcohol lipidoid compound with two different tails.

In another aspect, novel aminoalcohol lipidoid compounds for drugdelivery may be prepared by reacting a polyamine with a terminalepoxide.

wherein, R₁ represents alkyl chains of varying lengths, while R₂ throughR₄ generally represent various combinations of alkyl chains, polyamines,and hydrogen atoms. Reactions are set up by adding [N−1] equivalents ofepoxide to polyamine (where N is the number of 2° amines plus 2× numberof 1° amines in the polyamine starting material). This generates amixture enriched in compounds with [N−1] tails. Typically, thesecompounds are a mixture of various constitutional isomers, are usuallyisolable by chromatography on silica gel; the identity and purity of theproducts may be confirmed through ¹H/¹³C NMR spectroscopy and/or byMALDI-MS (with 2,5-dihydroxybenzoic acid matrix). As described herein,the epoxide, the amine, or both the epoxide and the amine may bestereochemically pure.

These inventive lipidoid compounds are also particularly useful in theadministration of polynucleotides. The aminoalcohol lipidoid compoundsof the present invention are amenable to combinatorial synthesis andscreening to generate libraries of compounds for use as nonviral drugdelivery agents. The inventive compounds may be used for other purposesas well such as coatings, additives, materials, and excipients.

In one aspect, the present invention provides a novel aminoalcohollipidoid compound of the formula:

as described herein. In another aspect, the present invention provides anovel aminoalcohol lipidoid compound of the formula:

as described herein.

In one aspect, the present invention provides novel aminoalcohollipidoid compounds based upon reacting a polyamine with a suitableterminal epoxide as described herein. In certain embodiments, thepolyamine is “amine 111” of the formula:

In certain embodiments, the polyamine is “amine 200” of the formula:

In certain embodiments, the polyamine is “amine 205” of the formula:

In certain embodiments, the polyamine is “amine 96” of the formula:

Materials based on amine 96 are generated through systematic variationaround the amine 96 core structure (see Example 15, Part 2).Aminoalcohol lipidoid compounds based upon amine 111 resulted fromperforming MALDI-MS analyses on the products of the amine 111 andepoxide reaction (see Example 14, Part 1).

In one aspect of the invention, the inventive aminoalcohol lipidoidcompounds are combined with an agent to be delivered to a cell or asubject to form microparticles, nanoparticles, liposomes, or micelles.The agent to be delivered by the particles, liposomes, or micelles maybe in the form of a gas, liquid, or solid, and the agent may be apolynucleotide, protein, peptide, or small molecule. The inventiveaminoalcohol lipidoid compounds may be combined with other aminoalcohollipidoid compounds, polymers (synthetic or natural), surfactants,cholesterol, carbohydrates, proteins, lipids, etc. to form theparticles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

The invention also provides methods of preparing the inventiveaminoalcohol lipidoid compounds. One or more equivalents of an amine areallowed to react with one or more equivalents of an epoxide-terminatedcompound under suitable conditions to form an aminoalcohol lipidoidcompound of the present invention. In certain embodiments, all the aminogroups of the amine are fully reacted with the epoxide-terminatedcompound to form tertiary amines. In other embodiments, all the aminogroups of the amine are not fully reacted with the epoxide-terminatedcompound to form tertiary amines thereby resulting in primary orsecondary amines in the aminoalcohol lipidoid compound. These primary orsecondary amines are left as is or may be reacted with anotherelectrophile such as a different epoxide-terminated compound. As will beappreciated by one skilled in the art, reacting an amine with less thanexcess of epoxide-terminated compound will result in a plurality ofdifferent aminoalcohol lipidoid compounds with various numbers of tails.Certain amines may be fully functionalized with two epoxide-derivedcompound tails while other molecules will not be completelyfunctionalized with epoxide-derived compound tails. For example, adiamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30°C.-100° C., preferably at approximately 50° C.-90° C. The preparedaminoalcohol lipidoid compounds may be optionally purified. For example,the mixture of aminoalcohol lipidoid compounds may be purified to yieldan aminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

The invention also provides libraries of aminoalcohol lipidoid compoundsprepared by the inventive methods. These aminoalcohol lipidoid compoundsmay be prepared and/or screened using high-throughput techniquesinvolving liquid handlers, robots, microtiter plates, computers, etc. Incertain embodiments, the aminoalcohol lipidoid compounds are screenedfor their ability to transfect polynucleotides or other agents (e.g.,proteins, peptides, small molecules) into the cell.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The “enantiomeric excess” of a substance is a measure of how pure adesired enantiomer is relative to the undesired enantiomer. Enantiomericexcess is defined as the absolute difference between the mole fractionof each enantiomer which is most often expressed as a percentenantiomeric excess. For mixtures of diastereomers, there are analogousdefinitions and uses for “diastereomeric excess” and percentdiastereomeric excess.

For example, a sample with 70% of R isomer and 30% of S will have anenantiomeric excess of 40%. This can also be thought of as a mixture of40% pure R with 60% of a racemic mixture (which contributes 30% R and30% S to the overall composition).

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In certain embodiments, a protectinggroup reacts selectively in good yield to give a protected substratethat is stable to the projected reactions; the protecting group shouldbe selectively removable in good yield by readily available, preferablynon-toxic reagents that do not attack the other functional groups; theprotecting group forms an easily separable derivative (more preferablywithout the generation of new stereogenic centers); and the protectinggroup has a minimum of additional functionality to avoid further sitesof reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbonprotecting groups may be utilized. Hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl,4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl,4-methoxytetrahydrothiopyranyl S,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described in Protective Groups in OrganicSynthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. Furthermore, this invention is not intended to be limitedin any manner by the permissible substituents of organic compounds.Combinations of substituents and variables envisioned by this inventionare preferably those that result in the formation of stable compoundsuseful in the treatment of diseases or disorders. The term “stable”, asused herein, preferably refers to compounds which possess stabilitysufficient to allow manufacture and which maintain the integrity of thecompound for a sufficient period of time to be detected and preferablyfor a sufficient period of time to be useful for the purposes detailedherein.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term“alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl,”“alkynyl,” and the like. Furthermore, as used herein, the terms “alkyl,”“alkenyl,” “alkynyl,” and the like encompass both substituted andunsubstituted groups. In certain embodiments, as used herein, “loweralkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched or unbranched) having 1-6 carbonatoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

The term “alkyl” as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. Examples of alkyl radicals include, but are not limitedto, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

The term “alkenyl” denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. Alkenyl groups include, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

The term “alkynyl” as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Representative alkynyl groups includeethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term “alkoxy,” or “thioalkyl” as used herein refers to an alkylgroup, as previously defined, attached to the parent molecule through anoxygen atom or through a sulfur atom. In certain embodiments, the alkyl,alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms. Incertain other embodiments, the alkyl, alkenyl, and alkynyl groupscontain 1-10 aliphatic carbon atoms. In yet other embodiments, thealkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4aliphatic carbon atoms. Examples of alkoxy, include but are not limitedto, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy,neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are notlimited to, methylthio, ethylthio, propylthio, isopropylthio,n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′,wherein R′ is aliphatic, as defined herein. In certain embodiments, thealiphatic group contains 1-20 aliphatic carbon atoms. In certain otherembodiments, the aliphatic group contains 1-10 aliphatic carbon atoms.In yet other embodiments, the aliphatic group employed in the inventioncontain 1-8 aliphatic carbon atoms. In still other embodiments, thealiphatic group contains 1-6 aliphatic carbon atoms. In yet otherembodiments, the aliphatic group contains 1-4 aliphatic carbon atoms.Examples of alkylamino groups include, but are not limited to,methylamino, ethylamino, n-propylamino, iso-propylamino,cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino,n-pentylamino, hexylamino, cyclohexylamino, and the like.

The term “carboxylic acid” as used herein refers to a group of formula—CO₂H.

The term “dialkylamino” refers to a group having the structure —NRR′,wherein R and R′ are each an aliphatic group, as defined herein. R andR′ may be the same or different in an dialkyamino moiety. In certainembodiments, the aliphatic groups contains 1-20 aliphatic carbon atoms.In certain other embodiments, the aliphatic groups contains 1-10aliphatic carbon atoms. In yet other embodiments, the aliphatic groupsemployed in the invention contain 1-8 aliphatic carbon atoms. In stillother embodiments, the aliphatic groups contains 1-6 aliphatic carbonatoms. In yet other embodiments, the aliphatic groups contains 1-4aliphatic carbon atoms. Examples of dialkylamino groups include, but arenot limited to, dimethylamino, methyl ethylamino, diethylamino,methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino,di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino,di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino,di(cyclohexyl)amino, and the like. In certain embodiments, R and R′ arelinked to form a cyclic structure. The resulting cyclic structure may bearomatic or non-aromatic. Examples of cyclic diaminoalkyl groupsinclude, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl,morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

In general, the terms “aryl” and “heteroaryl,” as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substituents recited for aliphatic moieties, orfor other moieties as disclosed herein, resulting in the formation of astable compound. In certain embodiments of the present invention, “aryl”refers to a mono- or bicyclic carbocyclic ring system having one or twoaromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. In certainembodiments of the present invention, the term “heteroaryl,” as usedherein, refers to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from S, O, and N; zero, one, ortwo ring atoms are additional heteroatoms independently selected from S,O, and N; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups can beunsubstituted or substituted, wherein substitution includes replacementof one, two, three, or more of the hydrogen atoms thereon independentlywith any one or more of the following moieties including, but notlimited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “cycloalkyl,” as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic, or hetercyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x))₂; —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “heteroaliphatic,” as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “heterocycloalkyl” or “heterocycle,” as used herein, refers toa non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto a benzene ring. Representative heterocycles include, but are notlimited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Incertain embodiments, a “substituted heterocycloalkyl or heterocycle”group is utilized and as used herein, refers to a heterocycloalkyl orheterocycle group, as defined above, substituted by the independentreplacement of one, two or three of the hydrogen atoms thereon with butare not limited to aliphatic; heteroaliphatic; aryl; heteroaryl;arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F;—Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples which aredescribed herein.

“Carbocycle”: The term “carbocycle,” as used herein, refers to anaromatic or non-aromatic ring in which each atom of the ring is a carbonatom.

“Independently selected”: The term “independently selected” is usedherein to indicate that the R groups can be identical or different.

“Labeled”: As used herein, the term “labeled” is intended to mean that acompound has at least one element, isotope, or chemical compoundattached to enable the detection of the compound. In general, labelstypically fall into three classes: a) isotopic labels, which may beradioactive or heavy isotopes, including, but not limited to, ²H, ³H,³²P, ³⁵S, ⁶⁷Ga, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb and ¹⁸⁶Re;b) immune labels, which may be antibodies or antigens, which may bebound to enzymes (such as horseradish peroxidase) that producedetectable agents; and c) colored, luminescent, phosphorescent, orfluorescent dyes. It will be appreciated that the labels may beincorporated into the compound at any position that does not interferewith the biological activity or characteristic of the compound that isbeing detected. In certain embodiments of the invention, photoaffinitylabeling is utilized for the direct elucidation of intermolecularinteractions in biological systems. A variety of known photophores canbe employed, most relying on photoconversion of diazo compounds, azides,or diazirines to nitrenes or carbenes (See, Bayley, H., PhotogeneratedReagents in Biochemistry and Molecular Biology (1983), Elsevier,Amsterdam.), the entire contents of which are hereby incorporated byreference. In certain embodiments of the invention, the photoaffinitylabels employed are o-, m- and p-azidobenzoyls, substituted with one ormore halogen moieties, including, but not limited to4-azido-2,3,5,6-tetrafluorobenzoic acid.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “heterocyclic,” as used herein, refers to a non-aromaticpartially unsaturated or fully saturated 3- to 10-membered ring system,which includes single rings of 3 to 8 atoms in size and bi- andtri-cyclic ring systems which may include aromatic six-membered aryl oraromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic rings include those having from one to three heteroatomsindependently selected from oxygen, sulfur, and nitrogen, in which thenitrogen and sulfur heteroatoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized.

The term “heteroaryl”, as used herein, refers to a cyclic aromaticradical having from five to ten ring atoms of which one ring atom isselected from sulfur, oxygen, and nitrogen; zero, one, or two ring atomsare additional heteroatoms independently selected from sulfur, oxygen,and nitrogen; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine,4-(2-chlorophenyl)piperazine, 4-(2-cyanophenyl)piperazine,4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine,4-(2-fluorophenyl)piperazine, 4-(2-hydroxyethyl)piperazine,4-(2-methoxyethyl)piperazine, 4-(2-methoxyphenyl)piperazine,4-(2-methylphenyl)piperazine, 4-(2-methylthiophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-phenylethyl)piperazine, 4-(2-pyridyl)piperazine,4-(2-pyrimidinyl)piperazine, 4-(2,3-dimethylphenyl)piperazine,4-(2,4-difluorophenyl) piperazine, 4-(2,4-dimethoxyphenyl)piperazine,4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine,4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine,4-(3-methylphenyl)piperazine, 4-(3-trifluoromethylphenyl)piperazine,4-(3,4-dichlorophenyl)piperazine, 4-3,4-dimethoxyphenyl)piperazine,4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term “substituted,” whether preceded by the term “optionally” ornot, and “substituent,” as used herein, refer to the ability, asappreciated by one skilled in this art, to change one functional groupfor another functional group provided that the valency of all atoms ismaintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents may also be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted with fluorine at one ormore positions).

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). An animal may be a transgenic animal.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc. In certain embodiments, an aninoalcohollipidoid compound is associated with a polynucleotide throughelectrostatic interactions.

“Biocompatible”: The term “biocompatible,” as used herein is intended todescribe compounds that are not toxic to cells. Compounds are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and their administration in vivo does notinduce inflammation or other such adverse effects.

“Biodegradable”: As used herein, “biodegradable” compounds are thosethat, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant toxic effect on the cells (i.e.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain embodiments,the chemical reactions relied upon to break down the biodegradablecompounds are uncatalyzed.

“Effective amount”: In general, the “effective amount” of an activeagent or composition refers to the amount necessary to elicit thedesired biological response. As will be appreciated by those of ordinaryskill in this art, the effective amount of an agent or device may varydepending on such factors as the desired biological endpoint, the agentto be delivered, the composition of the encapsulating matrix, the targettissue, etc. For example, the effective amount of microparticlescontaining an antigen to be delivered to immunize an individual is theamount that results in an immune response sufficient to preventinfection with an organism having the administered antigen.

“Peptide” or “protein”: According to the present invention, a “peptide”or “protein” comprises a string of at least three amino acids linkedtogether by peptide bonds. The terms “protein” and “peptide” may be usedinterchangeably. Peptide may refer to an individual peptide or acollection of peptides. Inventive peptides preferably contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in aninventive peptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. In certainembodiments, the modifications of the peptide lead to a more stablepeptide (e.g., greater half-life in vivo). These modifications mayinclude cyclization of the peptide, the incorporation of D-amino acids,etc. None of the modifications should substantially interfere with thedesired biological activity of the peptide.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotiderefers to a polymer of nucleotides. Typically, a polynucleotidecomprises at least three nucleotides. The polymer may include naturalnucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

“Small molecule”: As used herein, the term “small molecule” refers toorganic compounds, whether naturally-occurring or artificially created(e.g., via chemical synthesis) that have relatively low molecular weightand that are not proteins, polypeptides, or nucleic acids. Typically,small molecules have a molecular weight of less than about 1500 g/mol.In certain embodiments, the small molecule is uncharged. In certainembodiments, the small molecule is negatively charged. Also, smallmolecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, and sulfonamides.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts a general synthetic scheme for preparing aminoalcohollipidoids by combining amines and epoxides, and reacting them atapproximately 90° C.

FIG. 2 depicts exemplary amines containing between two and five aminefunctionalities and racemic epoxides of varying tails, unique functionalgroups and varying degrees of saturation that may be used for preparingaminoalcohol lipidoids.

FIG. 3 depicts characterization data of aminoalcohol lipidoids derivedfrom amine 114.

FIG. 4 depicts thin layer chromatography (TLC) plates of selectedcompounds from the aminoalcohol lipidoid library. Plate “A” depictsfully substituted amines, while Plate “B” depicts n−1 substitutedamines.

FIG. 5 depicts Firefly luciferase knockdown results relative tountreated cells from complexing RNA (50 ng) with various aminoalcohollipidoids (at various wt/wt ratios) and incubated with HeLa cells.

FIG. 6 depicts luciferase gene delivery results (Luciferase Expression,“RLU”) in HepG2 cells for various epoxide lipidoid compounds in 10%serum and 0.3 μg of DNA per well.

FIG. 7 depicts Factor VII Knockdown in vivo results and characterizationof C57BL/6 mice 48 hours after administration (via tail vein injectionat a volume of 0.01 ml/g) of (a) phosphate buffered saline; (b) 1.75mg/kg entrapped siRNA in lipidoid formulation; and (c) 4 mg/kg entrappedsiRNA in lipidoid formulation.

FIG. 8 depicts luciferase knockdown results (measured by relativeluceriferase expression, % control) for a library of aminoalcohollipidoid compounds wherein the ratio of aminoalcohol lipidoid compoundto siRNA is 2.5:1 (w/w).

FIG. 9 depicts the luciferase knockdown results (measured by relativeluceriferase expression, % control) for fifteen aminoalcohol lipidoidcompounds having >90% knockdown wherein the ratio of aminoalcohollipidoid compound to siRNA is 2.5:1 (w/w).

FIG. 10 depicts: (a) dose (mg/kg) response results (measured by FactorVII knockdown in mice by aminoalcohol lipidoid C14-110 36 hourspost-injection) wherein the ratio of aminoalcohol lipidoid compound tosiRNA is 10:1 (w/w), the ratio of aminoalcohol lipidoidcompound:cholesterol:PEG is 42:48:10, and 44% entrapment of 91 nmparticles; and (b) average of % BW change.

FIG. 11 depicts in vitro screening results as luciferase knockdown inHeLa cells by 25 epoxide-based lipidoids at 5:1 w/w ratio.

FIG. 12 depicts in vivo screening results as Factor VII knockdown 48hours post-injection of formulated epoxide lipidoids in mice.

FIG. 13a depicts dose response results for C16-96B as Factor VIIknockdown 48 hours post-injection of formulation C16-96-B in mice.

FIG. 13b depicts corresponding mice body weight loss and/or gain duringthe experimental that provided the results in FIG. 13.

FIG. 14a depicts dose response results for C14-110B as Factor VIIknockdown 72 hours post-injection of formulation C14-110-B in mice.

FIG. 14b depicts corresponding mice body weight loss and/or gain duringthe experiment that provided the results in FIG. 14 a.

FIG. 15 depicts C16-96-B formulation optimization as Factor VIIknockdown 48 hours post-injection of formulation C16-96-B in mice at 1mg/kg dose.

FIG. 16 depicts C16-96-B dose response as Factor VII knockdown 48 hourspost-injection of formulation C16-96-B in mice.

FIG. 17a depicts additional in vivo screening and discovery of C12-200and/or C12-205 as Factor VII knockdown 48 hours post-injection offormulated lipidoids in mice at 0.25 mg/kg dose.

FIG. 17b depicts additional in vivo screening and discovery of C12-200and/or C12-205 and corresponding mice body weight loss and/or gainduring the experiment that provided the results in FIG. 17 a.

FIG. 18a depicts dose response results for C12-200 and/or C12-205 andND98 comparison as Factor VII knockdown 48 hours post-injection offormulated C12-200 and/or C12-205 in mice.

FIG. 18b depicts corresponding mice body weight loss and/or gain duringthe experiment that provided the results in FIG. 18 a.

FIG. 19 depicts formulation optimization of C12-200 and/or C12-205 asFactor VII knockdown 48 hours post-injection of formulated C12-200and/or C12-205 in mice at 0.01 mg/kg dose.

FIG. 20a depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) ofthe crude reaction mixture of technical grade 111 amine and C12 epoxide.

FIG. 20b depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) ofthe “purified” product from the crude reaction mixture of technicalgrade 111 amine and C12 epoxide (from FIG. 20a ).

FIG. 21a depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) ofthe crude reaction mixture of technical grade 111 amine and C12 epoxide.

FIG. 21b depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) ofthe “purified” product from the crude reaction mixture of technicalgrade 111 amine and C12 epoxide (from FIG. 21a ).

FIG. 22 depicts an ¹H NMR (400 MHz) spectrum of C12-200 and/or C12-205(chloroform, room temperature).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides novel aminoalcohol lipidoid compounds anddrug delivery systems based on the use of such aminoalcohol lipidoidcompounds. The system may be used in the pharmaceutical/drug deliveryarts to delivery polynucleotides, proteins, small molecules, peptides,antigen, drugs, etc. to a patient, tissue, organ, cell, etc. These novelcompounds may also be used as materials for coating, additives,excipients, materials, bioengineering, etc.

The aminoalcohol lipidoid compounds of the present invention provide forseveral different uses in the drug delivery art. The amine-containingportion of the aminoalcohol lipidoid compounds may be used to complexpolynucleotides, thereby enhancing the delivery of polynucleotide andpreventing their degradation. The aminoalcohol lipidoid compounds mayalso be used in the formation of picoparticles, nanoparticles,microparticles, liposomes, and micelles containing the agent to bedelivered. Preferably, the aminoalcohol lipidoid compounds arebiocompatible and biodegradable, and the formed particles are alsobiodegradable and biocompatible and may be used to provide controlled,sustained release of the agent to be delivered. These lipidoids andtheir corresponding particles may also be responsive to pH changes giventhat these lipidoids are protonated at lower pH. The lipidoids may alsoact as proton sponges in the delivery of an agent to a cell to causeendosome lysis.

1. Aminoalcohol Lipidoid Compounds

The aminoalcohol lipidoid compounds of the present invention areaminoalcohol lipidoid compounds containing primary, secondary, tertiary,and/or quaternary amines, and salts thereof. The amines may be cyclic oracyclic amines. In certain embodiments, the inventive aminoalcohollipidoid compounds are relatively non-cytotoxic. In another embodiment,the inventive aminoalcohol lipidoid compounds are biocompatible andbiodegradable. In certain embodiments, the aminoalcohol lipidoids of thepresent invention have pK_(a)s in the range of approximately 5.5 toapproximately 7.5, more preferably between approximately 6.0 andapproximately 7.0. In another embodiment, the aminoalcohol lipidoidcompounds may be designed to have a desired pK_(a) between approximately3.0 and approximately 9.0, or between approximately 5.0 andapproximately 8.0. The inventive aminoalcohol lipidoid compounds areparticularly attractive for drug delivery for several reasons: 1) theycontain amino groups for interacting with DNA, RNA, otherpolynucleotides, and other negatively charged agents, for buffering thepH, for causing endosomolysis, for protecting the agent to be delivered,etc.; 2) they can be synthesized from commercially available startingmaterials; and/or 3) they are pH responsive and can be engineered with adesired pK_(a).

In certain embodiments, the aminoalcohol lipidoid compound orcomposition containing aminoalcohol lipidoid compound(s), are thosederived from terminated epoxides of 14 carbons or greater coupled withmonomers of three or more amine functional groups. In certainembodiments, the composition containing an aminoalcohol lipidoidcompound is about 40-60% lipidoid, about 40-60% cholesterol, and about5-20% PEG. In certain embodiments, the composition containing anaminoalcohol lipidoid compound is about 50-60% lipidoid, about 40-50%cholesterol, and about 5-10% PEG. In certain embodiments, thecomposition containing an aminoalcohol lipidoid compound is 52%lipidoid, 48% cholesterol, and 10% PEG. In certain embodiments, thecomposition containing an aminoalcohol lipidoid is about 50-75%lipidoid, about 20-40% cholesterol, and about 1-10% PEG. In certainembodiments, the composition containing an aminoalcohol lipidoidcompound is about 60-70% lipidoid, about 25-35% cholesterol, and about5-10% PEG.

In certain embodiments, the aminoalcohol lipidoid compounds may beprepared by reacting an amine with a terminal epoxide or an aldehydeaccording to the following schemes.

In certain embodiments, the epoxide is stereochemically pure (e.g.,enantiomerically pure). In certain embodiments, the amine isstereochemically pure (e.g., enantiomerically pure). In certainembodiments, the lipidoid is prepared from the reductive amination of animine which derived from the condensation of an amine and an aldehyde.In certain embodiments, the aminoalcohol lipidoid compounds of thepresent invention are of one of the formulae:

wherein each occurrence of A, R₁, R₂, R₃, R₄, R_(B), R_(C), R_(D),R_(A), R_(F), m, p, x, and y are as defined herein. As will beappreciated by one of skill in the art, the amine may be reacted with anexcess of epoxide to form a fully functionalized aminoalcohol lipidoidcompound. Or, the lipidoid may have fewer epoxide-derived tails thanwhen fully functionalized. In certain embodiments, each

is independently

each

is independently

each

is independently

and each

is independently

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

A is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic C₂₋₂₀ alkylene, optionally interrupted by 1 or more heteroatomsindependently selected from O, S and N, or A is a substituted orunsubstituted, saturated or unsaturated 4-6-membered ring;

R₁ is hydrogen, a substituted, unsubstituted, branched or unbranchedC₁₋₂₀-aliphatic or a substituted, unsubstituted, branched or unbranchedC₁₋₂₀ heteroaliphatic, wherein at least one occurrence of R₁ ishydrogen;

R_(B), R_(C), and R_(D) are, independently, hydrogen, a substituted,unsubstituted, branched or unbranched C₁₋₂₀-aliphatic, or a substituted,unsubstituted, branched or unbranched C₁₋₂₀-heteroaliphatic or—CH₂CH(OH)R_(E);

R_(B) and R_(D) together may optionally form a cyclic structure;

R_(C) and R_(D) together may optionally form a cyclic structure; and

R_(E) is a substituted, unsubstituted, branched or unbranched C₁₋₂₀aliphatic or a substituted, unsubstituted, branched or unbranched C₁₋₂₀heteroaliphatic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, A is an unsubstituted, unbranched, and acyclicC₂₋₂₀ alkylene. In certain embodiments, A is a substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₂₋₂₀,alkylene, optionally interrupted by 1 or more nitrogen atoms. In certainembodiments, A is a substituted, unbranched, and acyclic C₂₋₁₀ alkylene,optionally interrupted, by 1 oxygen atom. In certain embodiments, A isof the formula

In certain embodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted, by1 or more oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2oxygen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive. In certainembodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2nitrogen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive.

In certain embodiments, A is selected from the following formulae:

In certain embodiments, R₁ is hydrogen. In certain embodiments, R₁ is anunsubstituted and unbranched, C₁₋₂₀-aliphatic or C₁₋₂₀ heteroaliphaticmoiety. In some embodiments, R₁ is an unsubstituted and unbranched,C₁₀₋₁₂-aliphatic group. In some embodiments, R₁ is

In some embodiments, R₁ is an unsubstituted and unbranched, C₁₃heteroaliphatic group. In some embodiments, R₁ is

In so embodiments, R₁ is an unsubstituted and unbranched, C₁₄heteroaliphatic group. In some embodiments, R₁ is

In certain embodiments, R₁ is selected from the following formulae:

In certain embodiments, R₁ is a C₁₋₂₀ alkenyl moiety, optionallysubstituted. In certain embodiments, R₁ is selected from the followingformulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, each

is independently

In certain embodiments, R₁ is:

In certain embodiments, R₁ is selected from the following formulae:

In certain embodiments, R₁ is selected from the following formulae:

In certain embodiments, R₁ is fluorinated. In certain embodiments R₁ isa fluorinated aliphatic moiety. In certain embodiments R₁ isperfluorinated. In certain embodiments R₁ is a perfluorinated aliphaticmoiety. In certain embodiments, R₁ is a perfluorinated C₁₋₂₀ alkylgroup. In certain embodiments, R₁ is selected from the followingformulae:

In certain embodiments, R₁ is selected from the following formulae:

In certain embodiments, R_(B) is hydrogen. In certain embodiments, R_(B)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(B) is C₁₋₆-alkyl. In certain embodiments R_(B) is methyl.In certain embodiments R_(B) is ethyl. In certain embodiments R_(B) ispropyl. In certain embodiments R_(B) is butyl. In certain embodiments,R_(B) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(B) is C₁₋₆-heteroalkyl. In certain embodiments,R_(B) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(C) is hydrogen. In certain embodiments, R_(C)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(C) is C₁₋₆-alkyl. In certain embodiments R_(C) is methyl.In certain embodiments R_(C) is ethyl. In certain embodiments R_(C) ispropyl. In certain embodiments R_(C) is butyl. In certain embodiments,R_(C) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(C) is C₁₋₆-heteroalkyl. In certain embodiments,R_(C) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(D) is hydrogen. In certain embodiments, R_(D)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(D) is C₁₋₆-alkyl. In certain embodiments R_(D) is methyl.In certain embodiments R_(D) is ethyl. In certain embodiments R_(D) ispropyl. In certain embodiments R_(D) is butyl. In certain embodiments,R_(D) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(D) is C₁₋₆-heteroalkyl. In certain embodiments,R_(D) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(B), R_(C), and R_(D) are all the same. Incertain embodiments, R_(B), R_(C), and R_(D) are all hydrogen or allC₁-C₆ alkyl. In certain embodiments, R_(B), R_(C), and R_(D) are allhydrogen. In certain embodiments, R_(B), R_(C), and R_(D) are all C₁-C₆alkyl. In certain embodiments, R_(B), R_(C), and R_(D) are allhydroxyalkyl. In certain embodiments, R_(B), R_(C), and R_(D) are allaminoalkyl. In certain embodiments, R_(B), R_(C), and R_(D) are hydrogenor methyl. In certain embodiments, at least two of R_(B), R_(C), andR_(D) are the same. In certain embodiments, R_(B), R_(C), and R_(D) areall different.

In certain embodiments, R_(E) is hydrogen. In certain embodiments, R_(E)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(E) is C₁₋₆-alkyl. In certain embodiments R_(E) is methyl.In certain embodiments R_(E) is ethyl. In certain embodiments R_(E) ispropyl. In certain embodiments R_(E) is butyl. In certain embodiments,R_(E) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(E) is C₆-heteroalkyl.

Particular exemplary compounds include:

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

A is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic C₂₋₂₀ alkylene, optionally interrupted by 1 or more heteroatomsindependently selected from O, S and N, or A is a substituted orunsubstituted, saturated or unsaturated 4-6-membered ring;

R₁ and R₂ are, independently, hydrogen, a substituted, unsubstituted,branched or unbranched C₁₋₂₀-aliphatic or a substituted, unsubstituted,branched or unbranched C₁₋₂₀ heteroaliphatic, wherein at least oneoccurrence of R₁ is hydrogen and at least one occurrence of R₂ ishydrogen;

R_(C) and R_(D) are, independently, hydrogen, a substituted,unsubstituted, branched or unbranched C₁₋₂₀-aliphatic, or a substituted,unsubstituted, branched or unbranched C₁₋₂₀-heteroaliphatic or—CH₂CH(OH)R_(E);

R_(C) and R_(D) together may optionally form a cyclic structure; and

R_(E) is a substituted, unsubstituted, branched or unbranched C₁₋₂₀aliphatic or a substituted, unsubstituted, branched or unbranched C₁₋₂₀heteroaliphatic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, A is an unsubstituted, unbranched, and acyclicC₂₋₂₀ alkylene. In certain embodiments, A is a substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₂₋₂₀ alkylene,optionally interrupted by 1 or more nitrogen atoms. In certainembodiments A is a substituted, unbranched, and acyclic C₂₋₁₀ alkylene,optionally interrupted by 1 oxygen atom. In certain embodiments, A is ofthe formula

In certain embodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2oxygen atoms. In certain embodiments, A is of the formula.

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive. In certainembodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2nitrogen atoms. In certain embodiments, A is of the formula,

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive.

In certain embodiments, A is selected from the following formulae:

In certain embodiments, R₁ and R₂ are hydrogen. In certain embodiments,R₁ and R₂ are, independently, an unsubstituted and unbranched,C₁₋₂₀-aliphatic or C₁₋₂₀ heteroaliphatic moiety. In some embodiments, R₁and R₂ are, independently, an unsubstituted and unbranchedC₁₀₋₁₂-aliphatic group. In some embodiments, R₁ and R₂ are

In some embodiments, R₁ and R₂ are, independently, an unsubstituted andunbranched, C₁₃ heteroaliphatic group. In some embodiments, R₁ and R₂are

In some embodiments, R₁ and R₂ are, independently, an unsubstituted andunbranched, C₁₄ heteroaliphatic group. In some embodiments, R₁ and R₂are

In certain embodiments, R₁ and R₂ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₂ are, a C₁₋₂₀ alkenyl moiety,optionally substituted. In certain embodiments, R₁ and R₂ are,independently, selected from the following formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₁ and R₂ are:

In certain embodiments, R₁ and R₃ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₂ are fluorinated. In certainembodiments R₁ and R₂ are a fluorinated aliphatic moiety. In certainembodiments R₁ and R₂ are perfluorinated. In certain embodiments R₁ andR₂ are a perfluorinated aliphatic moiety. In certain embodiments, R₁ andR₂ are a perfluorinated C₁₋₂₀ alkyl group. In certain embodiments, R₁and R₂ are selected from the following formulae:

In certain embodiments, R₁ and R₂ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₂ are both the same. In certainembodiments, each of R₁ and R₂ are independently hydrogen or C₁-C₆alkyl. In certain embodiments, R₁ and R₂ are both hydrogen. In certainembodiments, R₁ and R₂ are both C₁-C₆ alkyl. In certain embodiments, R₁and R₂ are both hydroxyalkyl. In certain embodiments, R₁ and R₂ are bothaminoalkyl. In certain embodiments, R₁ and R₂ are different.

In certain embodiments, R_(C) is hydrogen. In certain embodiments, R_(C)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(C) is C₁₋₆-alkyl. In certain embodiments R_(C) is methyl.In certain embodiments R_(C) is ethyl. In certain embodiments R_(C) ispropyl. In certain embodiments R_(C) is butyl. In certain embodiments,R_(C) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(C) is C₁₋₆-heteroalkyl. In certain embodiments,R_(C) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(D) is hydrogen. In certain embodiments, R_(D)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(D) is C₁₋₆-alkyl. In certain embodiments R_(D) is methyl.In certain embodiments R_(D) is ethyl. In certain embodiments R_(D) ispropyl. In certain embodiments R_(D) is butyl. In certain embodiments,R_(D) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(D) is C₁₋₆-heteroalkyl. In certain embodiments,R_(D) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(C) and R_(D) are both the same. In certainembodiments, each of R_(C) and R_(D) are independently hydrogen, orC₁-C₆ alkyl. In certain embodiments, R_(C) and R_(D) are both hydrogen.In certain embodiments, R_(C) and R_(D) are both C₁-C₆ alkyl. In certainembodiments, R_(C) and R_(D) are both hydroxyalkyl. In certainembodiments, R_(C) and R_(D) are both aminoalkyl. In certainembodiments, R_(C) and R_(D) are different.

In certain embodiments, R_(E) is hydrogen. In certain embodiments, R_(E)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(E) is C₁₋₆-alkyl. In certain embodiments R_(E) is methyl.In certain embodiments R_(E) is ethyl. In certain embodiments R_(E) ispropyl. In certain embodiments R_(E) is butyl. In certain embodiments,R_(E) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(E) is C₆-heteroalkyl.

Particular exemplary compounds include:

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

A is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic C₂₋₂₀ alkylene, optionally interrupted by 1 or more heteroatomsindependently selected from O, S and N, or A is a substituted orunsubstituted, saturated or unsaturated 4-6-membered ring;

R₁ and R₃ are, independently, hydrogen, a substituted, unsubstituted,branched or unbranched C₁₋₂₀-aliphatic or a substituted, unsubstituted,branched or unbranched C₁₋₂₀ heteroaliphatic, wherein at least oneoccurrence of R₁ is hydrogen and at least one occurrence of R₃ ishydrogen;

R_(B) and R_(D) are, independently, hydrogen, a substituted,unsubstituted, branched or unbranched C₁₋₂₀-aliphatic, or a substituted,unsubstituted, branched or unbranched C₁₋₂₀-heteroaliphatic or—CH₂CH(OH)R_(E);

R_(B) and R_(D) together may optionally form a cyclic structure; and

R_(E) is a substituted, unsubstituted, branched or unbranched C₁₋₂₀aliphatic or a substituted, unsubstituted, branched or unbranched C₁₋₂₀heteroaliphatic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, each

is independently

and each

is independently

In certain embodiments, A is an unsubstituted, unbranched, and acyclicC₂₋₂₀ alkylene. In certain embodiments, A is a substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₂₋₂₀ alkylene,optionally interrupted by 1 or more nitrogen atoms. In certainembodiments A is a substituted, unbranched, and acyclic C₂₋₁₀ alkylene,optionally interrupted by 1 oxygen atom. In certain embodiments, A is ofthe formula

In certain embodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2oxygen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive. In certainembodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2nitrogen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive.

In certain embodiments, A is selected from the following formulae:

In certain embodiments, R₁ and R₃ are hydrogen. In certain embodiments,R₁ and R₃ are, independently, an unsubstituted and unbranched,C₁₋₂₀-aliphatic or C₁₋₂₀ heteroaliphatic moiety. In some embodiments, R₁and R₃ are, independently, an unsubstituted and unbranched,C₁₀₋₁₂-aliphatic group. In some embodiments, R₁ and R₃ are

In some embodiments, R₁ and R₃ are, independently, an unsubstituted andunbranched, C₁₃ heteroaliphatic group. In some embodiments, R₁ and R₃are

In some embodiments, R₁ and R₃ are, independently, an unsubstituted andunbranched, C₁₄ heteroaliphatic group. In some embodiments, R₁ and R₃are

In certain embodiments, R₁ and R₃ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₃ are, a C₁₋₂₀ alkenyl moiety,optionally substituted. In certain embodiments, R₁ and R₃ are,independently, selected from the following formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₁ and R₃ are:

In certain embodiments, R₁ and R₃ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₃ are fluorinated. In certainembodiments R₁ and R₃ are a fluorinated aliphatic moiety. In certainembodiments R₁ and R₃ are perfluorinated. In certain embodiments R₁ andR₃ are a perfluorinated aliphatic moiety. In certain embodiments, R₁ andR₃ are a perfluorinated C₁₋₂₀ alkyl group. In certain embodiments, R₁and R₃ are selected from the following formulae:

In certain embodiments, R₁ and R₃ are, independently, selected from thefollowing formulae:

In certain embodiments, R₁ and R₃ are both the same. In certainembodiments, each of R₁ and R₃ are independently hydrogen, or C₁-C₆alkyl. In certain embodiments, R₁ and R₃ are both hydrogen. In certainembodiments, R₁ and R₃ are both C₁-C₆ alkyl. In certain embodiments, R₁and R₃ are both hydroxyalkyl. In certain embodiments, R₁ and R₃ are bothaminoalkyl. In certain embodiments, R₁ and R₃ are different.

In certain embodiments, R_(B) is hydrogen. In certain embodiments, R_(B)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(B) is C₁₋₆-alkyl. In certain embodiments R_(B) is methyl.In certain embodiments R_(B) is ethyl. In certain embodiments R_(B) ispropyl. In certain embodiments R_(B) is butyl. In certain embodiments,R_(B) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(B) is C₁₋₆-heteroalkyl. In certain embodiments,R_(B) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(D) is hydrogen. In certain embodiments, R_(D)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(D) is C₁₋₆-alkyl. In certain embodiments R_(D) is methyl.In certain embodiments R_(D) is ethyl. In certain embodiments R_(D) ispropyl. In certain embodiments R_(D) is butyl. In certain embodiments,R_(D) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(D) is C₁₋₆-heteroalkyl. In certain embodiments,R_(D) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(B) and R_(D) are both the same. In certainembodiments, each of R_(B) and R_(D) are independently hydrogen, orC₁-C₆ alkyl. In certain embodiments, R_(B) and R_(D) are both hydrogen.In certain embodiments, R_(B) and R_(D) are both C₁-C₆ alkyl. In certainembodiments, R_(B) and R_(D) are both hydroxyalkyl. In certainembodiments, R_(B) and R_(D) are both aminoalkyl. In certainembodiments, R_(B) and R_(D) are different.

In certain embodiments, R_(E) is hydrogen. In certain embodiments, R_(E)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(E) is C₁₋₆-alkyl. In certain embodiments R_(E) is methyl.In certain embodiments R_(E) is ethyl. In certain embodiments R_(E) ispropyl. In certain embodiments R_(E) is butyl. In certain embodiments,R_(E) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(E) is C₆-heteroalkyl.

Particular exemplary compounds include:

In certain embodiments, each

is independently

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

A is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic C₂₋₂₀ alkylene, optionally interrupted by 1 or more heteroatomsindependently selected from O, S and N, or A is a substituted orunsubstituted, saturated or unsaturated 4-6-membered ring;

R₁, R₂, and R₃ are, independently, hydrogen, a substituted,unsubstituted, branched or unbranched C₁₋₂₀-aliphatic or a substituted,unsubstituted, branched or unbranched C₁₋₂₀ heteroaliphatic, wherein atleast one occurrence of R₁ is hydrogen, at least one occurrence of R₂ ishydrogen and at least one occurrence of R₃ is hydrogen;

R_(D) is hydrogen, a substituted, unsubstituted, branched or unbranchedC₁₋₂₀-aliphatic, or a substituted, unsubstituted, branched or unbranchedC₁₋₂₀-heteroaliphatic or —CH₂CH(OH)R_(E); and

R_(E) is a substituted, unsubstituted, branched or unbranched C₁₋₂₀aliphatic or a substituted, unsubstituted, branched or unbranched C₁₋₂₀heteroaliphatic; or a pharmaceutically acceptable salt thereof.

In certain embodiments, each

is independently

each

is independently

and each

is independently

In certain embodiments, A is an unsubstituted, unbranched, and acyclicC₂₋₂₀ alkylene. In certain embodiments, A is a substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₂₋₂₀ alkylene,optionally interrupted by 1 or more nitrogen atoms. In certainembodiments A is a substituted, unbranched, and acyclic C₂₋₁₀ alkylene,optionally interrupted by 1 oxygen atom. In certain embodiments, A is ofthe formula

In certain embodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2oxygen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive. In certainembodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2nitrogen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive.

In certain embodiments, A is selected from the following formulae:

In certain embodiments, R₁, R₂ and R₃ are hydrogen. In certainembodiments, R₁, R₂ and R₃ are, independently, an unsubstituted andunbranched, C₁₋₂₀-aliphatic or C₁₋₂₀ heteroaliphatic moiety. In someembodiments, R₁, R₂ and R₃ are, independently, an unsubstituted andunbranched, C₁₂-aliphatic group. In some embodiments, R₁, R₂ and R₃ are

In some embodiments, R₁, R₂ and R₃ are, independently, an unsubstitutedand unbranched, C₁₃ heteroaliphatic group. In some embodiments, R₁, R₂and R₃ are

In some embodiments, R₁, R₂ and R₃ are, independently, an unsubstitutedand unbranched, C₁₄ heteroaliphatic group. In some embodiments, R₁, R₂and R₃ are

In certain embodiments, R₁, R₂ and R₃ are, independently, selected fromthe following formulae:

In certain embodiments, R₁, R₂, and R₃ are, a C₁₋₂₀ alkenyl moiety,optionally substituted. In certain embodiments, R₁, R₂, and R₃ are,independently, selected from the following formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₁, R₂, and R₃ are:

In certain embodiments, R₁, R₂, and R₃ are, independently, selected fromthe following formulae:

In certain embodiments, R₁, R₂ and R₃ are fluorinated. In certainembodiments R₁, R₂ and R₃ are a fluorinated aliphatic moiety. In certainembodiments R₁, R₂ and R₃ are perfluorinated. In certain embodiments R₁,R₂ and R₃ are a perfluorinated aliphatic moiety. In certain embodiments,R₁, R₂ and R₃ are a perfluorinated C₁₋₂₀ alkyl group. In certainembodiments, R₁, R₂ and R₃ are selected from the following formulae:

In certain embodiments, R₁, R₂, and R₃ are, independently, selected fromthe following formulae:

In certain embodiments, R₁, R₂, and R₃ are all the same. In certainembodiments, at least two of R₁, R₂, and R₃ are the same. In certainembodiments, R₁, R₂, and R₃ are all different.

In certain embodiments, R_(D) is hydrogen. In certain embodiments, R_(D)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(D) is C₁₋₆-alkyl. In certain embodiments R_(D) is methyl.In certain embodiments R_(D) is ethyl. In certain embodiments R_(D) ispropyl. In certain embodiments R_(D) is butyl. In certain embodiments,R_(D) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(D) is C₁₋₆-heteroalkyl. In certain embodiments,R_(D) is —CH₂CH(OH)R_(E).

In certain embodiments, R_(E) is hydrogen. In certain embodiments, R_(E)is an unsubstituted and unbranched C₁₋₂₀-aliphatic. In certainembodiments R_(E) is C₁₋₆-alkyl. In certain embodiments R_(E) is methyl.In certain embodiments R_(E) is ethyl. In certain embodiments R_(E) ispropyl. In certain embodiments R_(E) is butyl. In certain embodiments,R_(E) is an unsubstituted and unbranched C₁₋₂₀-heteroaliphatic. Incertain embodiments R_(E) is C₁₋₆-heteroalkyl.

Particular exemplary compounds include:

In certain embodiments, each

is independently

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

A is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic C₂₋₂₀ alkylene, optionally interrupted by 1 or more heteroatomsindependently selected from O, S and N, or A is a substituted orunsubstituted, saturated or unsaturated 4-6-membered ring; and

R₁, R₂, R₃, and R₄ are, independently, hydrogen, a substituted,unsubstituted, branched or unbranched C₁₋₂₀-aliphatic or a substituted,unsubstituted, branched or unbranched C₁₋₂₀ heteroaliphatic, wherein atleast one occurrence of R₁ is hydrogen, at least one occurrence of R₂ ishydrogen, at least one occurrence of R₃ is hydrogen and at least oneoccurrence of R₄ is hydrogen; or a pharmaceutically acceptable saltthereof.

In certain embodiments, each

is independently

each

is independently

each

is independently

and each

is independently

In certain embodiments, A is an unsubstituted, unbranched, and acyclicC₂₋₂₀ alkylene. In certain embodiments, A is a substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₂₋₂₀ alkylene,optionally interrupted by 1 or more nitrogen atoms. In certainembodiments A is a substituted, unbranched, and acyclic C₂₋₁₀ alkylene,optionally interrupted by 1 oxygen atom. In certain embodiments, A is ofthe formula

In certain embodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore oxygen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2oxygen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive. In certainembodiments, A is a substituted or unsubstituted, branched orunbranched, cyclic or acyclic C₂₋₂₀ alkylene, optionally interrupted by1 or more nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 1 ormore nitrogen atoms. In certain embodiments A is an unsubstituted,unbranched, and acyclic C₂₋₁₀ alkylene, optionally interrupted by 2nitrogen atoms. In certain embodiments, A is of the formula

In certain embodiments, A is of the formula

wherein n is an integer between 1 and 10, inclusive.

In certain embodiments, A is selected from the following formulae:

In certain embodiments, R₁, R₂, R₃ and R₄ are hydrogen. In certainembodiments, R₁, R₂, R₃ and R₄ are, independently, an unsubstituted andunbranched, C₁₋₂₀-aliphatic or C₁₋₂₀ heteroaliphatic moiety. In someembodiments, R₁, R₂, R₃ and R₄ are, independently, an unsubstituted andunbranched, C₁₂-aliphatic group. In some embodiments, R₁, R₂, R₃ and R₄are

In some embodiments, R₁, R₂, R₃ and R₄ are, independently, anunsubstituted and unbranched, C₁₃ heteroaliphatic group. In someembodiments, R₁, R₂, R₃ and R₄ are

In some embodiments, R₁, R₂, R₃ and R₄ are, independently, anunsubstituted and unbranched, C₁₄ heteroaliphatic group. In someembodiments, R₁, R₂, R₃ and R₄ are

In certain embodiments, R₁, R₂, R₃ and R₄ are, independently, selectedfrom the following formulae:

In certain embodiments, R₁, R₂, R₃, and R₄ are, a C₁₋₂₀ alkenyl moiety,optionally substituted. In certain embodiments, R₁, R₂, R₃, and R₄ are,independently, selected from the following formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₁, R₂, R₃, and R₄ are:

In certain embodiments, R₁, R₂, R₃, and R₄ are, independently, selectedfrom the following formulae:

In certain embodiments, R₁, R₂, R₃, and R₄ are fluorinated. In certainembodiments R₁, R₂, R₃, and R₄ are a fluorinated aliphatic moiety. Incertain embodiments R₁, R₂, R₃, and R₄ are perfluorinated. In certainembodiments R₁, R₂, R₃, and R₄ are a perfluorinated aliphatic moiety. Incertain embodiments, R₁, R₂, R₃, and R₄ are a perfluorinated C₁₋₂₀ alkylgroup. In certain embodiments, R₁, R₂, R₃, and R₄ are selected from thefollowing formulae:

In certain embodiments, R₁, R₂, R₃, and R₄ are, independently, selectedfrom the following formulae:

In certain embodiments, R₁, R₂, R₃, and R₄ are all the same. In certainembodiments, at least two of R₁, R₂, R₃, and R₄ are the same. In certainembodiments, at least three of R₁, R₂, R₃, and R₄ are the same. Incertain embodiments, R₁, R₂, R₃, and R₄ are all different.

Particular exemplary compounds include:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 120 with the epoxide-terminated compound C14. In certainembodiments, the aminoalcohol lipidoid compound C14-120 is one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 120 with the epoxide-terminated compound C16. In certainembodiments, the aminoalcohol lipidoid compound C16-120 is of one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 98 with the epoxide-terminated compound C14. In certainembodiments, the aminoalcohol lipidoid compound C14-98 is of one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 113 with the epoxide-terminated compound C14. In certainembodiments, the aminoalcohol lipidoid compound C14-113 is of one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 96 with the epoxide-terminated compound C18. In certainembodiments, the aminoalcohol lipidoid compound is of one of theformulae below:

In certain embodiments, each

p is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 96 with the epoxide-terminated compound C14. In certainembodiments, the aminoalcohol lipidoid compound C14-96 is of one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 110 with the epoxide-terminated compound C14. In certainembodiments, the aminoalcohol lipidoid compound C14-110 is of one of theformulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

p is an integer between 1 and 3, inclusive;

m is an integer between 1 and 3, inclusive;

R_(A) is hydrogen; substituted or unsubstituted, cyclic or acyclic,branched or unbranched C₁₋₂₀ aliphatic; substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic;substituted or unsubstituted aryl; substituted or unsubstitutedheteroaryl;

R_(F) is hydrogen; substituted or unsubstituted, cyclic or acyclic,branched or unbranched C₁₋₂₀ aliphatic; substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic;substituted or unsubstituted aryl; substituted or unsubstitutedheteroaryl;

each occurrence of R₅ is independently hydrogen; substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀aliphatic; substituted or unsubstituted, cyclic or acyclic, branched orunbranched C₁₋₂₀ heteroaliphatic; substituted or unsubstituted aryl; orsubstituted or unsubstituted heteroaryl;

wherein, at least one of R_(A), R_(F), R_(Y), and R_(Z) is

each occurrence of x is an integer between 1 and 10, inclusive;

each occurrence of y is an integer between 1 and 10, inclusive;

each occurrence of R_(Y) is hydrogen; substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ aliphatic; substitutedor unsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀heteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl;

each occurrence of R_(Z) is hydrogen; substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ aliphatic; substitutedor unsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀heteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl;

or a pharmaceutically acceptable salt thereof.

In certain embodiments, R_(A) is hydrogen. In certain embodiments, R_(A)is hydrogen. In certain embodiments, R_(A) is substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀aliphatic. In certain embodiments, R_(A) is C₁-C₆ aliphatic. In certainembodiments, R_(A) is C₁-C₆ alkyl. In certain embodiments, R_(A) issubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedC₁₋₂₀ heteroaliphatic. In certain embodiments, R_(A) is substituted orunsubstituted aryl. In certain embodiments, R_(A) is substituted orunsubstituted heteroaryl. In certain embodiments, R_(A) is

In certain embodiments, each

is independently

In certain embodiments, R_(A) is

In certain embodiments, R_(A) is

In certain embodiments R_(A) is

In certain embodiments R_(A) is

In certain embodiments R_(A) is

In certain embodiments, R_(F) is hydrogen. In certain embodiments, noR_(F) is hydrogen. In certain embodiments, R_(F) is substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀aliphatic. In certain embodiments, R_(F) is C₁-C₆ aliphatic. In certainembodiments, R_(F) is C₁-C₆ alkyl. In certain embodiments, R_(F) issubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedC₁₋₂₀ heteroaliphatic. In certain embodiments, R_(F) is substituted orunsubstituted aryl. In certain embodiments, R_(F) is substituted orunsubstituted heteroaryl. In certain embodiments, R_(F) is

In certain embodiments, each

In certain embodiments, each

In certain embodiments, R_(F) is

In certain embodiments, R_(F) is

In certain embodiments R_(F) is

In certain embodiments R_(F) is

In certain embodiments R_(F) is

In certain embodiments R_(F) is

In certain embodiments R_(F)

In certain embodiments, no R_(A) is hydrogen and no R_(F) is hydrogen.In certain embodiments, R_(A) is

and R_(F) is

In certain embodiments, R_(A) is

and R_(F) is

In certain embodiments, each

In certain embodiments, each

In certain embodiments, R_(A) is

and R_(F) is

In certain embodiments, each

In certain embodiments, each

In certain embodiments, R_(A) is

and R_(F) is

In certain embodiments, R_(A) is

and R_(F) is

In certain embodiments, each

In certain embodiments, each

In certain embodiments, m is 1. In certain embodiments, m is 2. Incertain embodiments, m is 3.

In certain embodiments, p is 1. In certain embodiments, p is 2. Incertain embodiments, p is 3.

In certain embodiments, R₅ is hydrogen. In certain embodiments, R₅ issubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedC₁₋₂₀ aliphatic. In certain embodiments, R₅ is C₈-C₁₆ aliphatic. Incertain embodiments, R₅ is C₈-C₁₆ alkyl. In some embodiments, R₅ is anunsubstituted and unbranched, C₁₀₋₁₂-aliphatic group. In someembodiments, R₅ is

In some embodiments, R₅ is

In some embodiments, R₅ is

In certain embodiments, R₅ is selected from the following formulae:

In certain embodiments, R₅ is a C₁₋₂₀ alkenyl moiety, optionallysubstituted. In certain embodiments, R₅ is selected from the followingformulae:

In certain embodiments, R₅ is substituted or unsubstituted, cyclic oracyclic, branched or unbranched C₁₋₂₀ heteroaliphatic. In someembodiments, R₅ is an unsubstituted and unbranched, C₁₃ heteroaliphaticgroup. In some embodiments, R₅ is an unsubstituted and unbranched, C₁₄heteroaliphatic group. In certain embodiments, R₅ is:

In certain embodiments, R₅ is, independently, selected from thefollowing formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₅ is substituted or unsubstituted aryl. Incertain embodiments, R₅ is or substituted or unsubstituted heteroaryl.

In certain embodiments, R₅ is fluorinated. In certain embodiments R₅ isa fluorinated aliphatic moiety. In certain embodiments R₅ isperfluorinated. In certain embodiments R₅ is a perfluorinated aliphaticmoiety. In certain embodiments, R₅ is a perfluorinated C₁₋₂₀ alkylgroup. In certain embodiments, R₅ is selected from the followingformulae:

In certain embodiments, R₅ is selected from the following formulae:

In certain embodiments, each R₅ is independently hydrogen, or C₁-C₆alkyl. In certain embodiments, each R₅ is hydrogen. In certainembodiments, R₁ and R₃ each R₅ is C₁-C₆ alkyl. In certain embodiments,each R₅ is hydroxyalkyl. In certain embodiments, each R₅ is aminoalkyl.In certain embodiments, two R₅ variables are the same. In certainembodiments, three R₅ variables are the same. In certain embodiments,each R₅ variable is different from the other.

In certain embodiments, x is 1. In certain embodiments, x is 2. Incertain embodiments, x is 3. In certain embodiments, x is 4. In certainembodiments, x is 5. In certain embodiments, x is 6. In certainembodiments, x is 7. In certain embodiments, x is 8. In certainembodiments, x is 9. In certain embodiments, x is 10.

In certain embodiments, y is 1. In certain embodiments, y is 2. Incertain embodiments, y is 3. In certain embodiments, y is 4. In certainembodiments, y is 5. In certain embodiments, y is 6. In certainembodiments, y is 7. In certain embodiments, y is 8. In certainembodiments, y is 9. In certain embodiments, y is 10.

In certain embodiments, x is 1 and y is 2. In certain embodiments, x is1 and y is 3. In certain embodiments, x is 1 and y is 4. In certainembodiments, x is 1 and y is 5. In certain embodiments, x is 2 and y is2. In certain embodiments, x is 2 and y is 3.

In certain embodiments, R_(Y) is hydrogen. In certain embodiments, R_(Y)is substituted or unsubstituted, cyclic or acyclic, branched orunbranched C₁₋₂₀ aliphatic. In certain embodiments, R_(Y) is C₁-C₆alkyl. In certain embodiments, R_(Y) is substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic. Incertain embodiments, R_(Y) is substituted or unsubstituted aryl. Incertain embodiments, R_(Y) is substituted or unsubstituted heteroaryl.In certain embodiments, R_(Y) is

In certain embodiments, each

is independently

In certain embodiments, R_(Y) is

In certain embodiments, R_(Z) is hydrogen. In certain embodiments, R_(Z)is substituted or unsubstituted, cyclic or acyclic, branched orunbranched C₁₋₂₀ aliphatic. In certain embodiments, R_(Y) is C₁-C₆alkyl. In certain embodiments, R_(Z) is substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic. Incertain embodiments, R_(Z) is substituted or unsubstituted aryl. Incertain embodiments, R_(Z) is substituted or unsubstituted heteroaryl.In certain embodiments, R_(Z) is

In certain embodiments, each

is independently

In certain embodiments, R_(Z) is

Particular exemplary compounds include:

In certain embodiments, the aminoalcohol lipidoid compounds of thepresent invention comprises a mixture of formulae:

In certain embodiments, each

is independently

In certain embodiments, the aminoalcohol lipidoid compound of thepresent invention is of the formula:

wherein:

each occurrence of R_(A) is independently hydrogen; substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀aliphatic; substituted or unsubstituted, cyclic or acyclic, branched orunbranched C₁₋₂₀ heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl;

wherein at least one R_(A) is

each occurrence of R₅ is independently hydrogen; substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀aliphatic; substituted or unsubstituted, cyclic or acyclic, branched orunbranched C₁₋₂₀ heteroaliphatic; substituted or unsubstituted aryl; orsubstituted or unsubstituted heteroaryl;

each occurrence of x is an integer between 1 and 10, inclusive;

each occurrence of y is an integer between 1 and 10, inclusive;

or a pharmaceutically acceptable salt thereof.

In certain embodiments, R_(A) is hydrogen. In certain embodiments, noR_(A) is hydrogen. In certain embodiments, at least one R_(A) ishydrogen. In certain embodiments, R_(A) is substituted or unsubstituted,cyclic or acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic. Incertain embodiments, R_(A) is substituted or unsubstituted aryl. Incertain embodiments, R_(A) is substituted or unsubstituted heteroaryl.In certain embodiments, two R_(A)'s together may form a cyclicstructure. In certain embodiments, at least one R_(A) is

In certain embodiments, each

In certain embodiments, each

is independently

In certain embodiments, at least one R_(A) is

In certain embodiments, R_(A) is substituted or unsubstituted, cyclic oracyclic, branched or unbranched C₁₋₂₀ aliphatic. In certain embodiments,at least one R_(A) is an alkenyl group. In certain embodiments, at leastone R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is an alkynyl group. Incertain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is a substituted orunsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀heteroaliphatic group. In certain embodiments, at least one R_(A) is aheteroaliphatic group. In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, at least one R_(A) is

In certain embodiments, two R_(A) variables are the same. In certainembodiments, three R_(A) variables are the same. In certain embodiments,each R_(A) variable is different from the other.

In certain embodiments, R₅ is hydrogen. In certain embodiments, R₅ issubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedC₁₋₂₀ aliphatic. In certain embodiments, R₅ is C₈-C₁₆ aliphatic. Incertain embodiments, R₅ is C₈-C₁₆ alkyl. In some embodiments, R₅ is anunsubstituted and unbranched, C₁₀₋₁₂-aliphatic group. In someembodiments, R₅ is

In some embodiments, R₅ is

In some embodiments, R₅ is

In certain embodiments, R₅ is selected from the following formulae:

In certain embodiments, R₅ is a C₁₋₂₀ alkenyl moiety, optionallysubstituted. In certain embodiments, R₅ is selected from the followingformulae:

In certain embodiments, R₅ is substituted or unsubstituted, cyclic oracyclic, branched or unbranched C₁₋₂₀ heteroaliphatic. In someembodiments, R₅ is an unsubstituted and unbranched, C₁₃ heteroaliphaticgroup. In some embodiments, R₅ is an unsubstituted and unbranched, C₁₄heteroaliphatic group. In certain embodiments, R₅ is:

In certain embodiments, R₅ is, independently, selected from thefollowing formulae:

It will be appreciated by one of ordinary skill in the art that theabove substituents may have multiple sites of unsaturation, and could beso at any position within the substituent.

In certain embodiments, R₅ is substituted or unsubstituted aryl. Incertain embodiments, R₅ is or substituted or unsubstituted heteroaryl.In certain embodiments, R₅ is

In certain embodiments, each

is independently

In certain embodiments, R₅ is

In certain embodiments, R₅ is fluorinated. In certain embodiments R₅ isa fluorinated aliphatic moiety. In certain embodiments R₅ isperfluorinated. In certain embodiments R₅ is a perfluorinated aliphaticmoiety. In certain embodiments, R₅ is a perfluorinated C₁₋₂₀ alkylgroup. In certain embodiments, R₅ is selected from the followingformulae:

In certain embodiments, R₅ is selected from the following formulae:

In certain embodiments, each R₅ is independently hydrogen, or C₁-C₆alkyl. In certain embodiments, each R₅ is hydrogen. In certainembodiments, R₁ and R₃ each R₅ is C₁-C₆ alkyl. In certain embodiments,each R₅ is hydroxyalkyl. In certain embodiments, each R₅ is aminoalkyl.In certain embodiments, two R₅ variables are the same. In certainembodiments, three R₅ variables are the same. In certain embodiments,each R₅ variable is different from the other.

In certain embodiments, x is 1. In certain embodiments, x is 2. Incertain embodiments, x is 3. In certain embodiments, x is 4. In certainembodiments, x is 5. In certain embodiments, x is 6. In certainembodiments, x is 7. In certain embodiments, x is 8. In certainembodiments, x is 9. In certain embodiments, x is 10.

In certain embodiments, y is 1. In certain embodiments, y is 2. Incertain embodiments, y is 3. In certain embodiments, y is 4. In certainembodiments, y is 5. In certain embodiments, y is 6. In certainembodiments, y is 7. In certain embodiments, y is 8. In certainembodiments, y is 9. In certain embodiments, y is 10.

In certain embodiments, an aminoalcohol lipidoid compound or compositioncontaining aminoalcohol lipidoid compound(s) is prepared by reacting anamine of one of the formulae:

with an epoxide-containing compound of one of the formulae:

In certain embodiments, the epoxide-terminated compounds are of theformula:

In certain embodiments, the epoxide-containing compound is of theformula:

In certain embodiments, the epoxide contains one or more chiral centers,such as those shown below for amine C8b:

In certain embodiments, one equivalent of an amine is reacted with oneequivalent of an epoxide-terminated compound. In certain embodiments,one equivalent of an amine is reacted with one, two, three, four, five,six or more equivalents of an epoxide-terminated compound. In certainembodiments, the amount of epoxide-terminated compound is limiting toprevent the functionalization of all amino groups. The resultingaminoalcohol lipidoid or aminoalcohol lipidoid composition in theseinstances contain secondary amino groups and/or primary amino groups.Aminoalcohol lipidoid compounds having secondary amines are particularuseful in certain instances. In certain embodiments, amine-containingaminoalcohol lipidoid compounds that have not been fully functionalizedare further reacted with another electrophile (e.g., terminal epoxide,alkyl halide, etc.). Such further functionalization of the amines of theaminoalcohol lipidoid compound results in aminoalcohol lipidoidcompounds with different epoxide-compound derived tails. One, two,three, four, five, or more tails may be different from the other tailsof the aminoalcohol lipidoid compounds.

In certain embodiments, it will be appreciated by one skilled in the artthat the amine and the epoxide will react at the unsubstituted carbon ofthe epoxide resulting in an aminoalcohol lipidoid compound as shown inthe following schemes.

In certain embodiments, the epoxide is stereochemically pure (e.g.,enantiomerically pure). In certain embodiments, the amine isstereochemically pure (e.g., enantiomerically pure). In certainembodiments, the lipidoid is prepared from the reductive amination of animine which derived from the condensation of an amine and an aldehyde.The compounds of the invention can have an enantiomeric excess or adiastereomeric excess up to and including 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%,92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%,98.5%, 99%, 99.5%, or 100%.

In other embodiments, it will be appreciated by one skilled in the artthat the amine and the epoxide will react at the substituted carbon ofthe epoxide resulting in an aminoalcohol lipidoid compound as shown inthe following scheme.

While the above reaction may be less preferred, it is likely to occur atleast to some degree and may be more favored under certain reactionconditions. An aminoalcohol lipidoid compound may have amines that havereacted in one or both manners.

In certain embodiments, the amine and epoxide-terminated compound arereacted together neat. In other embodiments, the reaction is done in asolvent (e.g., THF, CH₂Cl₂, MeOH, EtOH, CHCl₃, hexanes, toluene,benzene, CCl₄, glyme, diethyl ether, etc.). In certain embodiments, thereaction mixture is heated. In certain embodiments, the reaction mixtureis heated to temperature ranging from 30° C.-100° C. In anotherembodiment, the reaction mixture is heated to approximately 90° C. Thereaction may also be catalyzed. For example, the reaction may becatalyzed by the addition of an acid, base, or metal (e.g., Lewis acid).The reaction may be allowed to proceed for hours, days, or weeks. Incertain embodiments, the reaction is allowed to proceed for 1-7 days. Incertain embodiments, the reactions were run from about 1 to about 3days. The resulting composition may be used with or withoutpurification. In certain embodiments, the lipidoids are subsequentlysubjected to an alkylation step (e.g., reaction with methyl iodide) toform quaternary amine salts. Optionally, various salt forms of thelipidoids may be prepared. In certain embodiments, the salts arepharmaceutically acceptable salts.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 200

with an epoxide-terminated compound. In certain embodiments, the amine200-derived aminoalcohol lipidoid compounds (i.e., C12-200) and itsvarious possible isomers are of the formulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 205

with an epoxide-terminated compound C12. In certain embodiments, theamine 205-derived aminoalcohol lipidoid compounds (i.e., C12-205) andits various possible isomers are of the formulae below:

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments, the aminoalcohol lipidoid is of the formula

In certain embodiments, the aminoalcohol lipidoid compound is of theformula

In certain embodiments, the aminoalcohol lipidoid compound is a mixtureof

In certain embodiments, each

is independently

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 96

with an epoxide-terminated compound C16. In certain embodiments, theamine 96-derived aminoalcohol lipidoid compounds (i.e., C16-96) and itsvarious possible isomers are of the formulae below:

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 210

with the epoxide-terminated compound C12. In a similar manner asillustrated above, one skilled in the art will readily be able todetermine the various possible 210-derived aminoalcohol lipidoidcompounds (i.e., C12-210) isomeric structures that are possible fromthis reaction.

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments the aminoalcohol lipidoid compound or compositioncontaining a mixture of aminoalcohol lipidoid compounds is prepared byreacting amine 220

with the epoxide-terminated compound C12. In a similar manner asillustrated above, one skilled in the art will readily be able todetermine the various possible 220-derived aminoalcohol lipidoidcompounds (i.e., C12-220) isomeric structures that are possible fromthis reaction.

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

In certain embodiments, the aminoalcohol lipidoid compound orcomposition containing a mixture of aminoalcohol lipidoid compounds isprepared by reacting amine 111

with the epoxide-terminated compound C12. In a similar manner asillustrated above, one skilled in the art will readily be able todetermine the various possible 111-derived aminoalcohol lipidoidcompounds (i.e., C12-111) isomeric structures that are possible fromthis reaction.

In certain embodiments the aminoalcohol lipidoid composition, is acomposition containing one or more of the above aminoalcohol lipidoidcompounds.

2. Synthesis of Aminoalcohol Lipidoid Compounds

The inventive aminoalcohol lipidoid compounds may be prepared by anymethod known in the art. Preferably the aminoalcohol lipidoid compoundsare prepared from commercially available starting materials, such asterminal-epoxide compounds, interior epoxide compounds, and amines. Inanother embodiment, the aminoalcohol lipidoid compounds are preparedfrom easily and/or inexpensively prepared starting materials. As wouldbe appreciated by one of skill in the art, the inventive aminoalcohollipidoid compounds can be prepared by total synthesis starting fromcommercially available starting materials. A particular aminoalcohollipidoid compound may be the desired final product of the synthesis, ora mixture of aminoalcohol lipidoid compounds may be the desired finalproduct.

In certain embodiments, the inventive aminoalcohol lipidoid compound isprepared by reacting an amine with an epoxide-terminated compound. Anexemplary reaction scheme is shown in FIG. 1.

Any amine containing between one, two, and five amine functionalities isuseful in preparing inventive aminoalcohol lipidoid compounds. Primaryamines useful in this invention include, but are not limited to,methylamine, ethylamine, isopropylamine, aniline, substituted anilines,ethanolamine, decylamine, undecylamine, dodecylamine, tetradecylamine,hexadecylamine, and octadecylamine. The amine may be a bis(primaryamine) including, but not limited to, ethylenediamine, 1,3diaminopropane, 1,4 diamino butane, 1,5 diaminopentane, 1,6diaminohexane, 2,2′(ethylenedioxy)bis(ethylamine). The amine may be abis(secondary amine). Secondary amines useful in this invention include,but are not limited to dipropylamine and methylpentylamine. The aminemay include both primary and secondary amines including, but not limitedto, (2-aminoethyl) ethanolamine, diethylenetriamine andtriethylenetetramine. Preferably, the amine is commercially available.In certain embodiments, the amine is stereochemically pure (e.g.,enantiomerically pure).

In certain embodiments, the amine used in the synthesis of theaminoalcohol lipidoid compound is of the formula:

Epoxide-terminated compounds that are useful in the present inventioninclude any epoxide-terminated compounds that are racemic orstereoisomers thereof, all of varying chain lengths and feature uniquefunctional groups having varying degrees of saturation. In certainembodiments, the epoxide is stereochemically pure (e.g.,enantiomerically pure). In certain embodiments, the epoxide contains oneor more chiral centers. In certain embodiments, the epoxide-terminatedcompounds are of the formula:

In certain embodiments, the epoxide-terminated compounds are of theformula:

In certain embodiments, the epoxide-terminated compounds are of theformula:

In certain embodiments, the epoxide contains one or more chiral centers,such as those shown below:

In certain embodiments, the enantiomeric epoxide

is resolved from the racemic mixture of epoxides using hydrolytickinetic resolution (HKR) catalyzed with the (R,R)-HKR catalyst of theformula:

In further embodiments, the enantiomeric epoxide

is resolved from the racemic mixture of epoxides using hydrolytickinetic resolution (HKR) catalyzed with the (S,S)-HKR catalyst of theformula:

In certain embodiments, the aminoalcohol lipidois of the invention areprepared from a process comprising steps of:(a) converting the epoxide primary alcohol of the formula:

into the corresponding protected primary alcohol derivative of theformula:

(b) reacting the protected primary alcohol derivative of the formula:

with a carbon-based nucleophile to produce the secondary alcohol of theformula:

(c) converting the secondary alcohol of the formula:

into the corresponding protected secondary alcohol derivative of theformula

(d) deprotecting the protected secondary alcohol derivative of theformula

into the corresponding primary alcohol of the formula

(e) oxidizing the primary alcohol of the formula

into the corresponding aldehyde of the formula

(f) condensing the aldehyde of the formula:

with an amine of the formula:

to produce an imine of the formula:

and (g) reducing an imine of the formula:

to produce the corresponding amine of the formula:

wherein R₁ is hydrogen, a substituted, unsubstituted, branched orunbranched C₁₋₂₀-aliphatic or a substituted, unsubstituted, branched orunbranched C₁₋₂₀ heteroaliphatic, wherein at least one occurrence of R₁is hydrogen; R_(B), R_(C), and R_(D) are, independently, hydrogen, asubstituted, unsubstituted, branched or unbranched C₁₋₂₀-aliphatic, or asubstituted, unsubstituted, branched or unbranched C₁₋₂₀-heteroaliphaticor —CH₂CH(OH)R_(E); R_(B) and R_(D) together may optionally form acyclic structure; R_(C) and R_(D) together may optionally form a cyclicstructure; R_(E) is a substituted, unsubstituted, branched or unbranchedC₁₋₂₀ aliphatic or a substituted, unsubstituted, branched or unbranchedC₁₋₂₀ heteroaliphatic; and PG₁ and PG₂ are O-protecting groups asdescribed herein.In certain embodiments, the epoxide primary alcohol of step (a) is

and the amine of step (f) is

In certain embodiments, the epoxide primary alcohol of step (a) is

and the amine of step (f) is

The chiral epoxides useful in the invention can be obtained from avariety of sources which are familiar to those skilled in the art oforganic synthesis. In some embodiments, the chiral epoxides useful inthe invention can be obtained commercially. In some embodiments, thechiral epoxides useful in the invention can be synthesized according tomethods known to those of skill in the art, such as, but not limited tothe Sharpless epoxidation of primary and secondary allylic alcohols into2,3-epoxyalcohols (Katsuki, et al., J. Am. Chem. Soc. 1980, 102, 5974;Hill, et al., Org. Syn., Coll. Vol. 7, p. 461 (1990); Vol. 63, p. 66(1985) and Katsuki, et al., Org. React. 1996, 48, 1-300; incorporatedherein by reference.) In some embodiments, the chiral epoxides useful inthe invention are obtained from the resolution of racemic epoxides. Insome embodiments, the chiral epoxides useful in the invention areobtained by the separation of enantiomers or diastereoisomers on chiralcolumns.

In certain embodiments, the reaction is performed neat without the useof a solvent. In other embodiments, a solvent is used for the reaction.Both or one of the starting amine or epoxide-terminated compound isdissolved in an organic solvent (e.g., THF, CH₂Cl₂, MeOH, EtOH, CHCl₃,hexanes, toluene, benzene, CCl₄, glyme, diethyl ether, etc.). Theresulting solutions are combined, and the reaction mixture is heated toyield the desired aminoalcohol lipidoid compound. In certainembodiments, the reaction mixture is heated to a temperature rangingfrom 25° C. to 100° C., preferably at approximately 90° C. The reactionmay also be catalyzed. For example, the reaction may be catalyzed by theaddition of an acid, base, or metal. The reagents may be allowed toreact for hours, days, or weeks. Preferably, the reaction is allowed toproceed from overnight (e.g., 8-12 hours) to 7 days.

The synthesized aminoalcohol lipidoid compounds may be purified by anytechnique known in the art including, but not limited to, precipitation,crystallization, chromatography, distillation, etc. In certainembodiments, the aminoalcohol lipidoid compound is purified throughrepeated precipitations in organic solvent (e.g., diethyl ether, hexane,etc.). In certain embodiments, the aminoalcohol lipidoid compound isisolated as a salt. The aminoalcohol lipidoid compound is reacted withan acid (e.g., an organic acid or inorganic acid) to form thecorresponding salt. In certain embodiments, the tertiary amine isalkylated to form a quaternary ammonium salt of the aminoalcohollipidoid compound. The tertiary amines may be alkylated with anyalkylating agent, for example, alkyl halides such as methyl iodide maybe used to from the quaternary amino groups. The anion associated withthe quaternary amine may be any organic or inorganic anion. Preferably,the anion is a pharmaceutically acceptable anion.

In certain embodiments, the reaction mixture results in a mixture ofisomers with varying numbers and positions of epoxide-derived compoundtails. Such mixtures of products or compounds may be used as is, or asingle isomer, or compound, may be purified from the reaction mixture.When an amine is not exhaustively alkylated, the resulting primary,secondary, or tertiary amines may be further reacted with anotheraminoalcohol lipidoid compound, epoxide-terminated compound, or otherelectrophile. The resulting aminoalcohol lipidoid compound may then beoptionally purified.

In certain embodiments, a desired aminoalcohol lipidoid compound isprepared by traditional total synthesis. In certain embodiments, acommercially available amine is the starting material. One or more aminogroups of the amine are optionally protected. The unprotected aminogroups are reacted with an epoxide-terminated compound. The product isoptionally purified. Protecting groups are removed, and the free aminogroups are optionally reacted with another aminoalcohol lipidoidcompound, epoxide-terminated compound, or other electrophile. Such asequence may be repeated depending on the desired complexity of theinventive product being prepared. The final product may then beoptionally purified.

In one embodiment, a library of different aminoalcohol lipidoidcompounds is prepared in parallel. A different amine and/orepoxide-terminated compound is added to each vial in a set of vials orto each well of a multi-well plate used to prepare the library. Thearray of reaction mixtures is incubated at a temperature and length oftime sufficient to allow formation of the aminoalcohol lipidoidcompounds to occur. In one embodiment, the vials are incubated atapproximately 90° C. overnight. In certain embodiments, the vials areincubated from 1 to 7 days at approximately 90° C. In certainembodiments, the vials are incubated from 3 to 4 days at approximately90° C. In certain embodiments, the vials are incubated from 1 to 2 daysat approximately 90° C. The aminoalcohol lipidoid compounds may then beisolated and purified using techniques known in the art. Theaminoalcohol lipidoid compounds may then be screened usinghigh-throughput techniques to identify aminoalcohol lipidoid compoundswith a desired characteristic (e.g., solubility in water, solubility atdifferent pH, ability to bind polynucleotides, ability to bind heparin,ability to bind small molecules, ability to bind protein, ability toform microparticles, ability to increase transfection efficiency, etc.).In certain embodiments the aminoalcohol lipidoid compounds may bescreened for properties or characteristics useful in gene therapy (e.g.,ability to bind polynucleotides, increase in transfection efficiency).

3. Polynucleotide Complexes

The ability of cationic compounds to interact with negatively chargedpolynucleotides through electrostatic interactions is well known.Cationic lipids such as Lipofectamine have been prepared and studied fortheir ability to complex and transfect polynucleotides. The interactionof the lipid with the polynucleotide is thought to at least partiallyprevent the degradation of the polynucleotide. By neutralizing thecharge on the backbone of the polynucleotide, the neutral orslightly-positively-charged complex is also able to more easily passthrough the hydrophobic membranes (e.g., cytoplasmic, lysosomal,endosomal, nuclear) of the cell. In certain embodiments, the complex isslightly positively charged. In certain embodiments, the complex has apositive ζ-potential, more preferably the ζ-potential is between 0 and+30.

The aminoalcohol lipidoid compounds of the present invention possesstertiary amines. Although these amines are hindered, they are availableto interact with a polynucleotide (e.g., DNA, RNA, synthetic analogs ofDNA and/or RNA, DNA/RNA hydrids, etc.). Polynucleotides or derivativesthereof are contacted with the inventive aminoalcohol lipidoid compoundsunder conditions suitable to form polynucleotide/lipidoid complexes. Thelipidoid is preferably at least partially protonated so as to form acomplex with the negatively charged polynucleotide. In certainembodiments, the polynucleotide/lipidoid complexes form particles thatare useful in the delivery of polynucleotides to cells. In certainembodiments, multiple aminoalcohol lipidoid molecules may be associatedwith a polynucleotide molecule. The complex may include 1-100aminoalcohol lipidoid molecules, 1-1000 aminoalcohol lipidoid molecules,10-1000 aminoalcohol lipidoid molecules, or 100-10,000 aminoalcohollipidoid molecules.

In certain embodiments, the complex may form a particle. In certainembodiments, the diameter of the particles ranges from 10-500micrometers. In certain embodiments, the diameter of the particlesranges from 10-1200 micrometers. In certain embodiments, the diameter ofthe particles ranges from 50-150 micrometers. In certain embodiments,the diameter of the particles ranges from 10-500 nm, more preferably thediameter of the particles ranges from 10-1200 nm, and most preferablyfrom 50-150 nm. The particles may be associated with a targeting agentas described below. In certain embodiments, the diameter of theparticles ranges from 10-500 pm, more preferably the diameter of theparticles ranges from 10-1200 pm, and most preferably from 50-150 pm.The particles may be associated with a targeting agent as describedbelow.

4. Polynucleotide

The polynucleotide to be complexed, encapsulated by the inventiveaminoalcohol lipidoid compounds, or included in a composition with theinventive aminoalcohol lipidoid compounds may be any nucleic acidincluding, but not limited to, RNA and DNA. In certain embodiments, thepolynucleotide is DNA. In certain embodiments, the polynucleotide isRNA.

In certain embodiments, the polynucleotide is an RNA that carries outRNA interference (RNAi). The phenomenon of RNAi is discussed in greaterdetail, for example, in the following references, each of which isincorporated herein by reference: Elbashir et al., 2001, Genes Dev.,15:188; Fire et al., 1998, Nature, 391:806; Tabara et al., 1999, Cell,99:123; Hammond et al., Nature, 2000, 404:293; Zamore et al., 2000,Cell, 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morrisand Rossi, 2006, Gene Ther., 13:553.

In certain embodiments, the polynucleotide is a dsRNA (double-strandedRNA).

In certain embodiments, the polynucleotide is an siRNA (shortinterfering RNA).

In certain embodiments, the polynucleotide is an shRNA (short hairpinRNA).

In certain embodiments, the polynucleotide is an miRNA (micro RNA).micro RNAs (miRNAs) are genomically encoded non-coding RNAs of about21-23 nucleotides in length that help regulate gene expression,particularly during development (see, e.g., Bartel, 2004, Cell, 116:281;Novina and Sharp, 2004, Nature, 430:161; and U.S. Patent Publication2005/0059005; also reviewed in Wang and Li, 2007, Front. Biosci.,12:3975; and Zhao, 2007, Trends Biochem. Sci., 32:189; each of which areincorporated herein by reference).

In certain embodiments, the polynucleotide is an antisense RNA.

In some embodiments, a dsRNA, siRNA, shRNA, miRNA and/or antisense RNAcan be designed and/or predicted using one or more of a large number ofavailable algorithms. To give but a few examples, the followingresources can be utilized to design and/or predict dsRNA, siRNA, shRNA,and/or miRNA: algorithms found at Alnylum Online, Dharmacon Online,OligoEngine Online, Molecula Online, Ambion Online, BioPredsi Online,RNAi Web Online, Chang Bioscience Online, Invitrogen Online, LentiWebOnline GenScript Online, Protocol Online; Reynolds et al., 2004, Nat.Biotechnol., 22:326; Naito et al., 2006, Nucleic Acids Res., 34:W448; Liet al., 2007, RNA, 13:1765; Yiu et al., 2005, Bioinformatics, 21:144;and Jia et al., 2006, BMC Bioinformatics, 7: 271; each of which isincorporated herein by reference).

The polynucleotides may be of any size or sequence, and they may besingle- or double-stranded. In certain embodiments, the polynucleotideis greater than 100 base pairs long. In certain embodiments, thepolynucleotide is greater than 1000 base pairs long and may be greaterthan 10,000 base pairs long. The polynucleotide is optionally purifiedand substantially pure. Preferably, the polynucleotide is greater than50% pure, more preferably greater than 75% pure, and most preferablygreater than 95% pure. The polynucleotide may be provided by any meansknown in the art. In certain embodiments, the polynucleotide has beenengineered using recombinant techniques (for a more detailed descriptionof these techniques, please see Ausubel et al. Current Protocols inMolecular Biology (John Wiley & Sons, Inc., New York, 1999); MolecularCloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, andManiatis (Cold Spring Harbor Laboratory Press: 1989); each of which isincorporated herein by reference). The polynucleotide may also beobtained from natural sources and purified from contaminating componentsfound normally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In certain embodiments, the polynucleotideis synthesized using standard solid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, etc.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesare preferably prepared using synthetic chemistry in vitro.

The polynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, etc.

The polynucleotide may be of any sequence. In certain embodiments, thepolynucleotide encodes a protein or peptide. The encoded proteins may beenzymes, structural proteins, receptors, soluble receptors, ionchannels, pharmaceutically active proteins, cytokines, interleukins,antibodies, antibody fragments, antigens, coagulation factors, albumin,growth factors, hormones, insulin, etc. The polynucleotide may alsocomprise regulatory regions to control the expression of a gene. Theseregulatory regions may include, but are not limited to, promoters,enhancer elements, repressor elements, TATA box, ribosomal bindingsites, stop site for transcription, etc. In certain embodiments, thepolynucleotide is not intended to encode a protein. For example, thepolynucleotide may be used to fix an error in the genome of the cellbeing transfected.

The polynucleotide may also be provided as an antisense agent or RNAinterference (RNAi) (Fire et al. Nature 391:806-811, 1998; incorporatedherein by reference). Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation (Crooke“Molecular mechanisms of action of antisense drugs” Biochim. Biophys.Acta 1489(1):31-44, 1999; Crooke “Evaluating the mechanism of action ofantiproliferative antisense drugs” Antisense Nucleic Acid Drug Dev.10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes313-314, 1999; each of which is incorporated herein by reference). Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix (i.e., triple helixformation) (Chan et al. J. Mol. Med. 75(4):267-282, 1997; incorporatedherein by reference).

In certain embodiments, the polynucleotide to be delivered comprises asequence encoding an antigenic peptide or protein. Nanoparticlescontaining these polynucleotides can be delivered to an individual toinduce an immunologic response sufficient to decrease the chance of asubsequent infection and/or lessen the symptoms associated with such aninfection. The polynucleotide of these vaccines may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health and can be found on theinternet (www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf,incorporated herein by reference; see also Allison Dev. Biol. Stand.92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; andPhillips et al. Vaccine 10:151-158, 1992; each of which is incorporatedherein by reference).

The antigenic protein or peptides encoded by the polynucleotide may bederived from such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

5. Particles

The aminoalcohol lipidoid compounds of the present invention may also beused to form drug delivery devices. The inventive aminoalcohol lipidoidcompounds may be used to encapsulate agents including polynucleotides,small molecules, proteins, peptides, metals, organometallic compounds,etc. The inventive aminoalcohol lipidoid compounds have severalproperties that make them particularly suitable in the preparation ofdrug delivery devices. These include: 1) the ability of the lipid tocomplex and “protect” labile agents; 2) the ability to buffer the pH inthe endosome; 3) the ability to act as a “proton sponge” and causeendosomolysis; and 4) the ability to neutralize the charge on negativelycharged agents. In certain embodiments, the aminoalcohol lipidoidcompounds are used to form particles containing the agent to bedelivered. These particles may include other materials such as proteins,carbohydrates, synthetic polymers (e.g., PEG, PLGA), and naturalpolymers.

In certain embodiments, the diameter of the particles range from between1 micrometer to 1,000 micrometers. In certain embodiments, the diameterof the particles range from between from 1 micrometer to 100micrometers. In certain embodiments, the diameter of the particles rangefrom between from 1 micrometer to 10 micrometers. In certainembodiments, the diameter of the particles range from between from 10micrometer to 100 micrometers. In certain embodiments, the diameter ofthe particles range from between from 100 micrometer to 1,000micrometers. In certain embodiments, the particles range from 1-5micrometers. In certain embodiments, the diameter of the particles rangefrom between 1 nm to 1,000 nm. In certain embodiments, the diameter ofthe particles range from between from 1 nm to 100 nm. In certainembodiments, the diameter of the particles range from between from 1 nmto 10 nm. In certain embodiments, the diameter of the particles rangefrom between from 10 nm to 100 nm. In certain embodiments, the diameterof the particles range from between from 100 nm to 1,000 nm. In certainembodiments, the particles range from 1-5 nm. In certain embodiments,the diameter of the particles range from between 1 pm to 1,000 pm. Incertain embodiments, the diameter of the particles range from betweenfrom 1 pm to 100 pm. In certain embodiments, the diameter of theparticles range from between from 1 pm to 10 pm. In certain embodiments,the diameter of the particles range from between from 10 pm to 100 pm.In certain embodiments, the diameter of the particles range from betweenfrom 100 pm to 1,000 pm. In certain embodiments, the particles rangefrom 1-5 pm.

6. Methods of Preparing Particles

The inventive particles may be prepared using any method known in thisart. These include, but are not limited to, spray drying, single anddouble emulsion solvent evaporation, solvent extraction, phaseseparation, simple and complex coacervation, and other methods wellknown to those of ordinary skill in the art. In certain embodiments,methods of preparing the particles are the double emulsion process andspray drying. The conditions used in preparing the particles may bealtered to yield particles of a desired size or property (e.g.,hydrophobicity, hydrophilicity, external morphology, “stickiness”,shape, etc.). The method of preparing the particle and the conditions(e.g., solvent, temperature, concentration, air flow rate, etc.) usedmay also depend on the agent being encapsulated and/or the compositionof the matrix.

Methods developed for making particles for delivery of encapsulatedagents are described in the literature (for example, please see Doubrow,M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRCPress, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987;Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of whichis incorporated herein by reference).

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve. The particle may also be coated. In certain embodiments,the particles are coated with a targeting agent. In other embodiments,the particles are coated to achieve desirable surface properties (e.g.,a particular charge).

7. Micelles and Liposomes

The aminoalcohol lipidoid compounds of the invention may be used toprepare micelles or liposomes. Many techniques for preparing micellesand liposomes are known in the art, and any method may be used with theinventive aminoalcohol lipidoid compounds to make micelles andliposomes. In addition, any agent including polynucleotides, smallmolecules, proteins, peptides, metals, organometallic compounds, etc.may be included in a micelle or liposome. Micelles and liposomes areparticularly useful in delivering hydrophobic agents such as hydrophobicsmall molecules.

In certain embodiments, liposomes (lipid or aminoalcohol lipidoidcompound vesicles) are formed through spontaneous assembly. In otherembodiments, liposomes are formed when thin lipid films or lipid cakesare hydrated and stacks of lipid crystalline bilayers become fluid andswell. The hydrated lipid sheets detach during agitation and self-closeto form large, multilamellar vesicles (LMV). This prevents interactionof water with the hydrocarbon core of the bilayers at the edges. Oncethese particles have formed, reducing the size of the particle can bemodified through input of sonic energy (sonication) or mechanical energy(extrusion). See Walde, P. “Preparation of Vesicles (Liposomes)” InEncylopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. AmericanScientific Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka etal. “Comparative Properties and Methods of Preparation of Lipid Vesicles(Liposomes)” Ann. Rev. Biophys. Bioeng. 9:467-508, 1980; each of whichis incorporated herein. The preparation of lipsomes involves preparingthe aminoalcohol lipidoid compounds for hydration, hydrating theaminoalcohol lipidoid compounds with agitation, and sizing the vesiclesto achieve a homogenous distribution of liposomes. Aminoalcohol lipidoidcompounds are first dissolved in an organic solvent to assure ahomogeneous mixture of aminoalcohol lipidoid compounds. The solvent isthen removed to form a lipidoid film. This film is thoroughly dried toremove residual organic solvent by placing the vial or flask on a vacuumpump overnight. Hydration of the lipidoid film/cake is accomplished byadding an aqueous medium to the container of dry lipidoid and agitatingthe mixture. Disruption of LMV suspensions using sonic energy typicallyproduces small unilamellar vesicles (SUV) with diameters in the range of15-50 nm. Lipid extrusion is a technique in which a lipid suspension isforced through a polycarbonate filter with a defined pore size to yieldparticles having a diameter near the pore size of the filter used.Extrusion through filters with 100 nm pores typically yields large,unilamellar vesicles (LUV) with a mean diameter of 120-140 nm.

In certain embodiments, the polynucleotide is an RNA molecule (e.g., anRNAi molecule). In other embodiments, the polynucleotide is a DNAmolecule. In certain embodiments, the aminoalcohol lipidoid is C14-120.In certain embodiments, the aminoalcohol lipidoid is C16-120. In certainembodiments, the aminoalcohol lipidoid is C14-98. In certainembodiments, the aminoalcohol lipidoid is C14-113. In certainembodiments, the aminoalcohol lipidoid is C18-96. In certainembodiments, the aminoalcohol lipidoid is C14-96. In certainembodiments, the aminoalcohol lipidoid is C14-110. In certainembodiments, the amount of aminoalcohol lipidoid compound in theliposome ranges from 30-80 mol %, preferably 40-70 mol %, morepreferably 60-70 mol %. These liposomes may be prepared using any methodknown in the art. In certain embodiments (e.g., liposomes containingRNAi molecules), the liposomes are prepared by lipid extrusion.

Certain aminoalcohol lipidoid compounds can spontaneously self assemblearound certain molecules, such as DNA and RNA, to form liposomes. Insome embodiments, the application is the delivery of polynucleotides.Use of these aminoalcohol lipidoid compounds allows for simple assemblyof liposomes without the need for additional steps or devices such as anextruder.

The following scientific papers described other methods for preparingliposomes and micelles: Narang et al. “Cationic Lipids with IncreasedDNA Binding Affinity for Nonviral Gene Transfer in Dividing andNondividing Cells” Bioconjugate Chem. 16:156-68, 2005; Hofland et al.“Formation of stable cationic lipid/DNA complexes for gene transfer”Proc. Natl. Acad. Sci. USA 93:7305-7309, July 1996; Byk et al.“Synthesis, Activity, and Structure-Activity Relationship Studies ofNovel Cationic Lipids for DNA Transfer” J. Med. Chem. 41(2):224-235,1998; Wu et al. “Cationic Lipid Polymerization as a Novel Approach forConstructing New DNA Delivery Agents” Bioconjugate Chem. 12:251-57,2001; Lukyanov et al. “Micelles from lipid derivatives of water-solublepolymers as delivery systems for poorly soluble drugs” Advanced DrugDelivery Reviews 56:1273-1289, 2004; Tranchant et al. “Physicochemicaloptimisation of plasmid delivery by cationic lipids” J. Gene Med.6:S24-S35, 2004; van Balen et al. “Liposome/Water Lipophilicity:Methods, Information Content, and Pharmaceutical Applications” MedicinalResearch Rev. 24(3):299-324, 2004; each of which is incorporated hereinby reference.

8. Agent

The agents to be delivered by the system of the present invention may betherapeutic, diagnostic, or prophylactic agents. Any chemical compoundto be administered to an individual may be delivered using the inventivecomplexes, picoparticles, nanoparticles, microparticles, micelles, orliposomes. The agent may be a small molecule, organometallic compound,nucleic acid, protein, peptide, polynucleotide, metal, an isotopicallylabeled chemical compound, drug, vaccine, immunological agent, etc.

In certain embodiments, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug. In certain embodiments, the drug is anantibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

In certain embodiments of the present invention, the agent to bedelivered may be a mixture of agents.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Prophylactic agents include, but are not limited to, antibiotics,nutritional supplements, and vaccines. Vaccines may comprise isolatedproteins or peptides, inactivated organisms and viruses, dead organismsand viruses, genetically altered organisms or viruses, and cellextracts. Prophylactic agents may be combined with interleukins,interferon, cytokines, and adjuvants such as cholera toxin, alum,Freund's adjuvant, etc. Prophylactic agents include antigens of suchbacterial organisms as Streptococccus pneumoniae, Haemophilusinfluenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

9. Targeting Agents

The inventive complexes, liposomes, micelles, microparticles,picoparticles and nanoparticles may be modified to include targetingagents since it is often desirable to target a particular cell,collection of cells, or tissue. A variety of targeting agents thatdirect pharmaceutical compositions to particular cells are known in theart (see, for example, Cotten et al. Methods Enzym. 217:618, 1993;incorporated herein by reference). The targeting agents may be includedthroughout the particle or may be only on the surface. The targetingagent may be a protein, peptide, carbohydrate, glycoprotein, lipid,small molecule, nucleic acids, etc. The targeting agent may be used totarget specific cells or tissues or may be used to promote endocytosisor phagocytosis of the particle. Examples of targeting agents include,but are not limited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelopeprotein of the human immunodeficiency virus (HIV), carbohydrates,receptor ligands, sialic acid, aptamers etc. If the targeting agent isincluded throughout the particle, the targeting agent may be included inthe mixture that is used to form the particles. If the targeting agentis only on the surface, the targeting agent may be associated with(i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, orother interactions) the formed particles using standard chemicaltechniques.

10. Pharmaceutical Compositions

Once the complexes, micelles, liposomes, or particles have beenprepared, they may be combined with one or more pharmaceuticalexcipients to form a pharmaceutical composition that is suitable toadminister to animals including humans. As would be appreciated by oneof skill in this art, the excipients may be chosen based on the route ofadministration as described below, the agent being delivered, timecourse of delivery of the agent, etc.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipidcomplexes), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1

Synthesis and Characterization of 1,2-aminoalcohols

These lipidoids were synthesized by combining amines and epoxides in aglass vial equipped with a stirbar and heated to 90° C., as shown inFIG. 1. The amines chosen contain between two and five aminefunctionalities, while the epoxides are racemic, of varying chainlengths and feature unique functional groups and varying degrees ofsaturation (FIG. 2). The reaction times varied from 24-72 hours at thistemperature. Mixtures generally remained clear throughout the reactionand became noticeably viscous as the reaction progressed. Upon cooling,many became waxy solids. The extent of the reaction could be controlledby the number of equivalents of epoxide added to the reaction mixture.For example, in the examples shown, amine 114 has a maximum of fivepoints for substitution. Addition of five equivalents of epoxide wouldyield an amine core with five alkane chains linked by a1,2-aminoalcohol. Addition of four equivalents of epoxide would yieldonly four chains linked by the same structure. This was verified by thinlayer chromatography (TLC), which showed primarily one product existingin the crude reaction mixtures set up as described.

To verify the identity of the molecules, a few test reactions were setup and purified by silica gel chromatography. The components of thecrude reaction mixture were separated and tested by NMR and massspectrometry. Again, in the case of amine 114, three products wereidentified: three, four and five tailed products. The molecular weightwas confirmed by mass spectrometry, and the structure was verified byNMR (FIG. 3, which shows characterization data of epoxide lipidoidsderived from amine 114). These isolated compounds were then used asstandards versus selected members of the library for TLC analysis.Reactions set up to fully substitute the amine had similar R_(f) andstaining profiles to the fully substituted standard. Reactions set up tooccupy n−1 positions of the amine had similar R_(f) and stainingprofiles to n−1 standard (FIG. 4).

Example 2

In Vitro Screening for RNA Delivery

Epoxide lipidoids were tested for their ability to deliver siRNA to aHeLa cell line that stably expresses both firefly and Renillaluciferase. Efficacy was determined by complexing the lipidoid withsiRNA specific for firefly luciferase, adding this mixture to cells andmeasuring the subsequent ratio of firefly to Renilla expression. Thisprocedure was performed in 96-well microtiter plates to enable highthroughput testing of the materials. In this assay, reduction of bothfirefly and Renilla expression indicates toxicity, while reduction ofonly firefly expression is an indication of specific knockdown due tosiRNA. Initial screening results of selected members of the library areshown in FIG. 5. Many members of this sampling showed some ability totransfect cells and give rise to some knockdown of firefly luciferase.Of these the best performers were generally lipidoids derived fromepoxides of 14 carbons or longer coupled with monomers of three or moreamine functional groups. A few show nearly complete ablation of fireflyexpression, even at the lowest dose of lipidoid tested.

Example 3

RNA Encapsulation Efficiency

Formulation for in vitro experiments is a simple mixing of RNA withlipidoid at a set ratio in buffer prior to addition to cells. In vivoformulation requires the addition of extra ingredients to facilitatecirculation throughout the body. To test the ability of these lipidoidsto form particles suitable for in vivo work, we followed a standardformulation procedure utilized in the lab. These particles consisted of42% lipidoid, 48% cholesterol and 10% PEG. After formation of theparticle, RNA was added and allowed to integrate with the complex. Theencapsulation efficiency was determined using a standard Ribogreenassay. As shown in the table below, these particles were on the order of100 nm after extrusion, with some achieving encapsulation efficiency ofover 90%.

Particle Size and Entrapment Efficiency of Selected Epoxide Lipidoids

Compound Size (nm) Entrapment (%) C14-120-B 95.2 92.75 C16-120-B 128.467.22 C14-98-B 126.9 44.84 C14-113-B 92.7 96.42

Example 4

HepG2 cells were seeded at a density of 15,000 cells per well intoopaque white 96-well plates (Corning-Costar, Kennebunk, Me.) 24 hoursprior to transfection to allow for growth and confluence. Workingdilutions of lipidoids were made in 25 mM sodium acetate (pH 5) at aconcentration of 0.5 mg/ml. For gene delivery experiments pCMV-Lucfirefly luciferase DNA (ElimBiopharmaceuticals, South San Francisco,Calif.) was used. Lipidoid:DNA complexes were formed by electrostaticinteraction between positively charged lipidoid molecules and negativelycharged nucleic acids. By varying the volume of lipidoid solution addedto a constant amount of DNA, varying weight:weight ratios of lipidoid toDNA were tested. Lipidoid solution (75 μl) was added to DNA solution (75μl) and mixed well. Mixtures were then incubated at room temperature for20 minutes to allow for complexation. These complexes (30 μl) were thenadded to serum containing medium (200 μl) and mixed well. Growth mediumwas then removed from the cells and lipidoid:DNA complex containingmedium was immediately added. Total DNA loading was 300 ug DNA per well.Lipofectamine 2000 transfection was performed as described by thevendor. Complexes were allowed to incubate with cells for 48 hours.Luciferase expression was then quantified by Bright-Glo assay (Promega,Madison, Wis.). Briefly, 48 hours post-transfection, the lipidoid:DNAcomplex containing growth medium was removed from cells using a12-channel aspirating wand. 200 ul of a 1:1 mixture of Bright-Gloreagent and non-phenol red containing DMEM was added to each well of the96-well plate with cells. After 10 minute incubation at roomtemperature, luminescence was measured using a luminometer. (n=3).Exemplary results are depicted in FIG. 6.

Example 5

Lipidoid-based siRNA formulations comprised lipidoid, cholesterol,polyethylene glycol-lipid (PEG-lipid) and siRNA. Stock solutions ofLipidoid, mPEG2000-DMG MW 2660 (synthesized by Alnylam), and cholesterolMW 387 (Sigma-Aldrich) were prepared in ethanol and mixed to yield amolar ratio of 42:10:48. Mixed lipids were added to 200 mM sodiumacetate buffer pH 5.2 to yield a solution containing 35% ethanol,resulting in spontaneous formation of empty lipidoid nanoparticles.Resulting nanoparticles were extruded through an 80 nm membrane (threepasses). siRNA in 35% ethanol and 50 mM sodium acetate pH 5.2 was addedto the nanoparticles at 10:1 (wt/wt) total lipids:siRNA and incubated at37° C. for 30 min. Ethanol removal and buffer exchange ofsiRNA-containing lipidoid nanoparticles was achieved by dialysis againstPBS using a 3,500 MWCO membrane. Particle size was determined using aMalvern Zetasizer NanoZS (Malvern). siRNA content and entrapmentefficiency was determined by Ribogreen assay.

C57BL/6 mice (Charles River Labs) received either saline or siRNA inlipidoid formulations via tail vein injection at a volume of 0.01 ml/g.Mice were dosed at either 1.75 or 4 mg/kg entrapped siRNA. At 48 hoursafter administration, animals were anesthetized by isofluoraneinhalation and blood was collected into serum separator tubes byretroorbital bleed. Serum levels of Factor VII protein were determinedin samples using a chromogenic assay (Biophen FVII, Aniara Corporation)according to the manufacturer's protocols. A standard curve wasgenerated using serum collected from saline-treated animals. Exemplaryresults are depicted in FIG. 7.

Example 6

In Vitro Screening of Epoxide Library

Compounds of the epoxide-based lipidoid library were synthesizedaccording to the procedures described herein. The compounds were thenscreened for siRNA delivery efficacy to a cancer cell line, using aHela-derived cell line genetically engineered to express luciferasereporter proteins. In these experiments, the ability of each material tofacilitate sequence-specific gene silencing was evaluated by comparisonof protein levels in treated groups to untreated controls. For eachcompound, delivery experiments were performed using varying weightratios of lipidoid:siRNA. In the original disclosure, knockdown resultsfor the entire library were shown. An abbreviated data set is shown inFIG. 11 demonstrating the results for the top 25 performing compounds inthe in vitro screen, including C16-96-B, C14-200 and/or C14-205, andC12-200 and/or C12-205.

Example 7

In Vivo Screening of Top Performing Epoxide Lipidoids

To test siRNA delivery efficacy in vivo, a mouse model for liverdelivery was used. Factor VII, a hepatocyte-specific blood clottingfactor, served as a model protein for knockdown studies. Once producedby hepatocytes, Factor VII is released into the bloodstream, and abaseline level of expression can be determined by simple blood draw andquantification of protein levels by colorimetric assay. By deliveringanti-Factor VII siRNA to hepatocytes, knockdown of this model proteincan be achieved and a percentage of silencing can be determined bycomparison to an untreated control.

Following the in vitro screen, compounds were purified as detailed inPart 1 (see Example 14). For in vivo testing, the compounds wereformulated with cholesterol and a PEG-lipid for serum stability andsiRNA packaging. In these experiments, lipidoids were formulated at a42:48:10 molar ratio of lipidoid:cholesterol:PEG. The weight ratio oftotal lipids (lipidoid+cholesterol+PEG) to siRNA was 10:1. After eachformulation, the particles were characterized for size and siRNAentrapment efficiency using dynamic light scattering and Ribogreenassay, respectively. The total dose of siRNA administered in the initialscreen varies from group to group due to the differences in entrapmentefficiency of the lipidoid particles. In all experiments, the dose ofsiRNA administered to each mouse is consistent according to body weight.The knockdown results from the in vivo screen are shown in FIG. 12. B1and B2 nomenclature signify different compounds visualized by TLC andisolated during purification. As shown, C14-11-B and C16-96-B were thelead compounds from the initial screen. It should be noted that whilesome compounds did not show efficacy in this screen, a simple adjustmentof formulation composition may greatly improve the results.

Example 8

Following the initial in vivo screening experiments, two compounds wereused to conduct a dose response. In these experiments and all subsequentexperiments, the siRNA dose is based on total siRNA content in theformulation, not entrapped siRNA. The dose response results are shown inFIG. 13a and FIG. 14a . In addition to Factor VII measurement, thechange in mouse body weight is recorded and a loss in weight isgenerally considered formulation induced toxicity (See FIG. 13b and FIG.14b ). Tables 1 and 2 tabulate the formulation parameters andcharacterization data from these experiments.

TABLE 1 Formulation parameters and characterization data for C16-96-Bdose response formulation Formulation Total Characterization LipidoidLipid:Chol:PEG Lipid:siRNA Entrapment Size C16-96-B 65:29:6 10:1 81%107.8 nm

TABLE 2 Formulation parameters and characterization data for C14-110-Bdose response formulation Formulation Total Characterization LipidoidLipid:Chol:PEG Lipid:siRNA Entrapment Size C14-110-B 42:48:10 10:1 44%115 nm

Example 9

After completing the dose response, C16-96-B was chosen for furtherinvestigation and optimization. In the next experiments, the percentcomposition of the formulations was varied to observe the effect ofcomposition on particle size, entrapment, and efficacy. The compositionsinvestigated are shown in Table 3. FIG. 15 shows the knockdown resultsfrom the formulations tested. Where the formulation in red was theprevious best, it is shown that efficacy can be improved by formulatingparticles at different compositions.

TABLE 3 Formulation parameters and characterization data for C16-96-Bformulation optimization experiment Formulation Parameters TotalFormulation Lipidoid Chol PEG Lipid:siRNA Entrapment 1 63 31 6 8.5:1 80%2 65 29 6 8.5:1 80% 3 67 27 6 8.5:1 80% 4 69 25 6 8.5:1 84% 5 71 23 68.5:1 85% 6 63 33 4 8.5:1 85% 7 65 31 4 8.5:1 85% 8 67 29 4 8.5:1 84% 969 27 4 8.5:1 83% 10 71 25 4 8.5:1 85%

Example 10

A second dose response was conducted with the new percent compositionparameters. The knockdown results and particleformulation/characteristics are shown in FIG. 16 and Table 4,respectively. By formulating at this composition, approximately 40%knockdown was achieved at a dose of 0.25 mg/kg. Using this result as thenew benchmark, the library was revisited and previously untestedmaterials were screened at 0.25 mg/kg in attempt to find other compoundswhich could give similar or better results.

TABLE 4 Formulation Total Characterization Lipidoid Lipid:Chol:PEGLipid:siRNA Entrapment Size C16-96-B 71:23:6 20:1 83% 205 nm

Example 11

In the revisited in vivo screen, compound C12-200 and/or C12-205 wasidentified as giving nearly complete silencing at a dose of 0.25 mg/kg.As depicted in FIG. 12, the slightly longer tailed version of thiscompound, C12-200 and/or C12-205 was previously identified as a highlyefficient delivery agent, showing complete silencing at a much higherdose of 7.5 mg/kg total siRNA. It is quite possible that this compoundcould facilitate complete silencing at much lower doses, but this hasnot yet been fully explored as the focus has been on C12-200 and/orC12-205. Following the discovery of the efficacy of this compound, thecharacterization experiments detailed in Part 1 (see Example 14) wereinitiated.

The knockdown and body weight change results of this screen are depictedin FIG. 17b , and Table 5 tabulates the formulation parameters andcharacteristics. FIG. 17a depicts knockdown results for a second batchof C16-96-B. In previous experiments, this compound had resulted inapproximately 40% knockdown at a dose of 0.25 mg/kg. From mass specanalysis, it was shown that this batch is two-tailed as opposed to themore efficacious three-tailed version that had been used in the previousstudies.

TABLE 5 Formulation parameters and characterization data for revisitedin vivo screening Formulation Parameters Total Entrapment SizeFormulation Lipid Chol PEG Lipid:siRNA (%) (nm) C14-96-B 73.1 21.3 5.68.5 87 81.7 C16-96-B 71.0 23.0 6.0 8.5 55 170.2 C12-200 and/or 45.0 45.59.5 8.5 36 167 C12-205 C18-62-B 52.7 39.1 8.2 8.5 86 227.4

Example 12

A low-dose response was performed on C12-200 and/or C12-205. Theknockdown and body weight loss results are shown in FIG. 18a and FIG.18b . The results show that efficient knockdown is achieved andextremely low doses of siRNA. In comparison to ND98, our previous bestcompound from the original lipidoid library, comparable knockdown can beachieved with 100-fold lower doses of siRNA. The formulation parametersand characterization data are shown in Table 6.

TABLE 6 Formulation parameters and characterization data for C12-200and/or C12-205 and ND98 formulations Formulation Parameters TotalEntrapment Size Formulation Lipid Chol PEG Lipid:siRNA (%) (nm) C12-200and/or 48.2 42.8 8.9 8.5 45 154.1 C12-205 ND98 42.0 48.0 10.0 8.5 9983.9

Example 13

To further improve delivery efficacy, the percent composition of theC12-200 and/or C12-205 formulation was modified incrementally. Theseformulations were screened at a dose of 0.01 mg/kg to identifyformulations which may perform better than the previous compositions.The results of these experiments are shown in FIG. 19 along with theformulation parameters and characteristics in Table 7. As expected, moreefficacious delivery can be achieved by tuning the composition of theformulation. This optimization work is currently ongoing, along withsynthesis of longer and shorter tailed versions of the C12-200 and/orC12-205 structure.

TABLE 7 Formulation parameters and characterization data for C12-200and/or C12-205formulations Formulation Parameters Total Entrapment SizeFormulation Lipid Chol PEG Lipid:siRNA (%) (nm) 1 65.0 25.0 10.0 8.5 0129 2 60.0 30.0 10.0 8.5 1 128 3 55.0 35.0 10.0 8.5 16 156 4 50.0 40.010.0 8.5 30 136 5 45.0 45.0 10.0 8.5 46 140 6 40.0 50.0 10.0 8.5 44 1687 35.0 55.0 10.0 8.5 40 154 8 50.0 45.0 5.0 8.5 29 157 9 45.0 50.0 5.08.5 34 154 10 40.0 55.0 5.0 8.5 27 159 11 35.0 60.0 5.0 8.5 34 155

Example 14

Part 1: Lipidoids Based on Amine 111

Amine 111 (tetraethylenepentamine, or TEPA) is represented as the linearpolyamine of the following structure:

The expected products of the reaction between amine 111 and the 12carbon terminal epoxide C12 are illustrated as follows.

This reaction was performed, and the crude reaction mixture wasseparated based on the assumption that the order of product elution frompolar silica gel would be: a) 7 tail (max substitution on 111 amine); b)6 tail isomers (the isomers corresponding to 6 epoxides having reactedwith the 111 amine); c) 5 tail isomers, and so on. It was expected thatthe MALDI-MS spectra of the crude reaction mixture would reveal peakscorresponding to the m/z ratios of these compounds (calculated [M+H⁺]for the expected 7 tail, 6 tail, and 5 tail products: 1481, 1295, and1111, respectively). Material was isolated from the crude reactionmixture that, based on TLC analysis, was assumed to be the mixture of 6tail isomers. This “purified” material performed quite well in the invivo anti-Factor VII transfection assay.

MALDI-MS spectra (see FIG. 20a ) of the crude reaction mixture and ofthe purified “6 tail” material suggested compounds (see FIG. 20b ).Technical grade tetraethlenepentamine (TEPA) is a mixture of compoundswith similar boiling points; some of these compounds are of thefollowing formulae:

Reaction of the C12 epoxide with these compounds accounts for most ofthe intense peaks in the MALDI mass spectra of the crude reactionmixture (FIG. 20a ). The m/z ratios observed for the “purified” material(FIG. 20b ) are consistent with amine 200 or 205 reacting with 5equivalents of epoxide (calculated m/z for [M+H⁺] 1137, found 1137) andwith amine 210 or 220 reacting with 4 equivalents of epoxide (calculatedm/z for [M+H⁺] 910, found 910). The structures of these compounds areillustrated as follows:

To determine if this result was reproducible, an epoxide ring openingreaction was performed using the C12 epoxide and two different batchesof amine 111. MALDI-MS was performed on each crude reaction mixture. Inthe reaction between the C12 epoxide and an older batch of 111 amine, anarray of compounds were observed that were also observed in the originalcrude reaction mixture (see FIG. 20a ). The MALDI spectrum of the crudereaction mixture using the C12 epoxide and a newer batch of amine 111(see FIG. 21a ) contains predominant peaks with m/z ratios of 1481(linear amine 111 and 7 epoxide tails) and 1137 (consistent with amine200 or 205 with 5 epoxide tails). The peak at m/z 910 in this spectrumwas small. This might be a result of batch to batch differences in the111 amine. Purification of the second reaction allowed isolation of ahighly pure sample of the m/z 1137 material; we have designated thismaterial “C12-200 and/or C12-205”. The ¹H NMR spectrum of C12-200 and/orC12-205 is consistent with the proposed structure (see FIG. 22).

We are developing a library of materials based on the reaction of amines200, 205, 210, and 220 and related amine structures with epoxides ofvarying length as depicted below:

These amines are being prepared in pure form using techniques familiarto one skilled in the art. We also propose a library of materialsderived from free-based commercially available 111 aminepentahydrochloride according to the following scheme:

the actual linear pentamine having the following structure:

Example 15

Part 2: Lipidoids Based on Amine 96

This Example describes the synthesis of a library of structures that arevariations of the amino alcohols lipidoid derived from the reaction ofamine 96 with a C16 epoxide as follows:

based upon the core 96 amine. First, variations at the position of themethyl group are achieved according to the following scheme:

by reacting the terminal epoxides with an assortment of commerciallyavailable amines as depicted below.

Based on this strategy, the resulting library would containapproximately 800 possible amino alcohols of varying structure.

Similar amine starting materials are available wherein the length of thecarbon chain between the two amines is longer or shorter than amine 96as depicted below.

A library of compounds resulting from the reaction of these amines withthe various terminal epoxides would provide an additional ˜700 aminoalcohol lipidoids.

A protection/deprotection synthetic strategy could provide multiplevariations, where the two core amines are functionalized with differentalkyl epoxides according to the following scheme:

This strategy could allows for substitution at one amine position with afunctional group other than an epoxide (e.g., alkyl halide,isothiocyanate, chloroformate, acid halide) generating two differentfunctional groups on the same amine core as follows:

The following is an exemplary scheme illustrates general syntheticprocedures to generate various compounds having two different functionalgroups on the same amine core as follows:

wherein, Y is an aryl, heteroaryl, alkyl group (unreactive withepoxides, isocyanates, isothiocyanates and/or alkyl halides); and Zrepresents fragment from isocyanate, isothiocyante, alkyl halide, havingthe following exemplary structures:

Various multi-step sequences could be used to introduce additionalhydroxyl groups near the amine core at positions different from thosegenerated through epoxide ring opening as follows:

Similar routes could provide the means to generate both hydroxyl groupsand additional unsaturation as follows:

Reductive amination as a first step after differential protection of theamine core provides access to a multitude of commercially availablealdehydes and perhaps a way to introduce multiple hydroxyl groupsthrough reductive amination using simple carbohydrates (a knownprocedure) as follows:

Example 16 Synthesis of1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200 and/or C12-205)

A 250 mL glass pressure vessel was charged with 2-decyloxirane (20.0grams, 109 mmoles), tetraethylenepentamine (Sigma-Aldrich technicalgrade, 2.93 grams, 15.5 mmoles) and a magnetic stir bar. The vessel wassealed and immersed in a silicone oil bath at 90° C. The reactionmixture was stirred vigorously for ˜72 hours at 90° C. The pressurevessel was then removed from the oil bath, allowed to cool to roomtemperature, then opened with caution. ˜9 grams of the resultingviscous, slightly yellow oil were purified via chromatography on silicagel (gradient elution from dichloromethane to 83.5:16.3:1.5dichloromethane/methanol/aqueous ammonium hydroxide). Fractionscontaining the desired compound were pooled and concentrated by rotaryevaporation. The resulting yellow oil was dissolved in ˜15 mL of ethylacetate; decolorizing charcoal was added to this mixture. The solutionwas warmed to 68° C. and then filtered through Celite; the filtrate wasconcentrated by rotary evaporation; residual solvent was removed underreduced pressure overnight affording ˜1.3 grams of a pale yellow viscousoil. The starting material may contain inseparable isomerN1-(2-aminoethyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine; andthe product may contain an inseparable isomer1,1′-(2-((2-hydroxydodecyl)(2-((2-hydroxydodecyl)(2-(4-(2-hydroxydodecyl)piperazin-1-yl)ethyl)amino)ethyl)-amino)-ethylazanediyl)-didodecan-2-ol.

Example 17

Amino Alcohol Lipidoids Prepared from Chiral Epoxides

Antimicrobial lipidoids (e.g., C12-200, C16-96) can be prepared byreacting lipophilic, racemic terminal epoxides with low molecular weightpolyamines. This approach is illustrated directly below with amine 200and a generic terminal epoxide. There are two problems with thisapproach that complicate the isolation of pure products: the use ofracemic epoxides, and addition of amines to the second carbon atom (C2)in the epoxide chain. In the following Examples, we report: a) theissues that may arise from the use of racemic epoxides can be avoidedthrough the use of stereochemically pure terminal epoxides; and b) sideproducts that may arise from additions at C2 of the epoxide can beavoided through an alternate synthetic route involving reductiveamination.

Reactions of Racemic Epoxides

The epoxides used in the initial library synthesis were purchased fromcommercial sources as racemic mixtures: each epoxide contained an equalproportion of the R and S enantiomer. Achiral amines are equally likelyto react with either stereoisomer. The effect of using racemic epoxidescan be illustrated by considering the simple case of the reactionbetween an amine with one reactive site (e.g., piperidine) and a racemicepoxide (illustrated directly below). In this case, two aminoalcohollipidoid products are generated: the R and S enantiomers. In theory,these products are separable through chromatography on a chiralstationary phase; in practice, developing and scaling up a method toperform this separation is difficult and expensive.

The situation becomes more complex when the starting amine has multiplereactive sites. For N reactive sites of an amine starting material,2^(N) stereoisomers are generated. For example, amine 200 (five reactivesites) reacts with a racemic epoxide generating 32 stereoisomers. In ourexperience, these products are inseparable. This issue can be resolvedby performing the reaction with epoxides that are stereochemically pure(e.g., a single enantiomer of an epoxide). This is illustrated directlybelow.

A few terminal epoxides are commercially available as singleenantiomers, but the cost of these compounds is prohibitive. Racemicepoxides can be resolved (separated into constituent enantiomers) byseveral means, including chromatography on a chiral stationary phase. Weresolved the epoxides using a chemical method known as hydrolytickinetic resolution (HKR). Efficient HKR of racemic epoxides can beachieved using a procedure described by Jacobsen (Schaus, et al., J. Am.Chem. Soc. 2002, 124, 1307-1315; which is incorporated herein byreference). The process is illustrated directly below. A chiral catalystand water are added to a solution containing the racemic epoxide. In thepresence of the chiral catalyst, the rate of hydrolysis of one epoxideenantiomer is much greater than the rate of hydrolysis for the otherenantiomer. This allows selective hydrolysis of the unwanted epoxideenantiomer (to a 1,2-diol). The 1,2-diol can be separated from theepoxide by removing the epoxide through distillation under reducedpressure.

Step 1. The Resolution of Epoxydodecane by HKR PGP-332C3

(R)-(+)-1,2-epoxydodecane

An oven-dried round bottom flask containing a magnetic stir bar wascharged with the (R,R)-HKR catalyst (Schaus, et al., J. Am. Chem. Soc.2002, 124, 1307-1315); CAS Number 176763-62-5, 1.31 g, 2.17 mmol.Dichloromethane (34 mL) and then glacial acetic acid (1.30 mL) wereadded to the flask. The resulting solution was stirred vigorously for1.5 h; during this time the color of the mixture changed from dark redto brown. The solvent was removed by rotary evaporation until thematerial appeared dry. 1,2-epoxydodecane (40.0 g, 217 mmol) thenisopropyl alcohol (reagent grade, 47 mL) were added to the flaskcontaining the oxidized catalyst and a magnetic stir bar. The flask wasimmersed in an ice bath. H₂O (2.15 mL, 119 mmol, 0.55 equiv relative toepoxide) was added dropwise to the stirred mixture. The flask was sealedwith a rubber septum and the solution was allowed to warm to roomtemperature. After stirring for 2 days, the reaction mixture was dilutedwith ˜200 mL of hexanes. The resulting solution was filtered throughpaper to remove the white precipitate (1,2-diol). The filtrate wasconcentrated by rotary evaporation. The resulting dark red oily liquidwas dissolved in ˜150 mL of hexanes and filtered in order to remove asubstantial amount of white crystalline precipitate (diol). The filtratewas transferred into a 250 mL round bottom flask and concentrated byrotary evaporation. The desired product was isolated by distillationunder vacuum (literature: 124° C./15 mm Hg). The desired product (14.3grams, 71.5% of theoretical yield) was collected as a clear oil. Theproduct was determined to be 100% ee by chiral chromatography of the2-napthylenethiol derivative.

Step 2. The Synthesis of (R)-C12-200

(R)-C12-200.

Amine 200 (640 mg, 2.97 mmol) and (R)-1,2-epoxydodecane (2.27 g, 12.1mmol) were added to a vial containing a magnetic stir bar. The vial wassealed and warmed on a 80° C. reaction block for 5 days. The reactionmixture was allowed to cool to room temperature, and the desired productwas isolated by chromatography on silica gel (gradient elution fromCH₂Cl₂ to 175:22:3 CH₂Cl₂/MeOH/NH₄OH (aq.)). Fractions were pooled andconcentrated affording (R)-C12-200 (665 mg) as a pale yellow oil. ¹H NMR(600 MHz, CDCl₃): δ 4.37 (br s, —OH, 4H), 3.63 (app. br s, 3H), 3.56(app. br s, 2H), 2.84-2.21 (m, 30H), 1.43-1.26 (m, 90H), 0.88 (t, J=7.0Hz, 15H); MALDI-TOF-MS m/z: calcd for C₇₀H₁₄₆N₅O₅ [M+H⁺] 1137.1, found1137.6.

Example 18 In Vivo Transfection with Chiral Amino Alcohol Lipidoids

Preliminary in vivo transfections using anti-Factor VII siRNA formulatedwith were performed using (R)-C12-200 and (S)-C12-200 in mice. At 0.01mg/kg siRNA dosing, approximately 50% reduction of systemic Factor VIIwas achieved using either the R or S forms of C12-200; there differencebetween these results and those obtained using C12-200 (the lipidoidprepared using amine 200 and racemic C12 epoxide) were insignificant.

Example 19 Synthesis of Amino Alcohol Lipidoids by the ReductiveAmination Approach

The first carbon atom in a terminal epoxide is the preferred site ofattack during nucleophilic addition. 2D-NMR analysis of amino alcohollipidoids shows that the majority of addition occurs at C1 of theepoxide, as illustrated directly below. Nevertheless, a trace amount ofaddition at C2 does occur. 2D-NMR analysis of compounds (S)-C12-205 and(R)-C12-200 suggest that roughly 10% of the lipid “tails” are the resultof amine attack at C2 of the epoxide. These regioisomeric tails arelikely distributed randomly throughout the entire population of lipidtails in the material. Efforts to limit this side reaction with theepoxides have not been successful. To avoid this side reaction, weproposed and executed an alternate synthetic strategy.

A retrosynthetic analysis of this strategy is presented directly below.The desired product is C (as illustrated directly below), from additionof amine A to C1 of epoxide B. D is the undesired constitutional isomerformed when amine A attacks C2 of epoxide B. Reductive amination ofaldehyde fragment E with amine A and a reducing agent (giving F),followed by removal of the protecting group on the secondary alcohol,should generate product C. This route does not generate undesiredstructure D. This approach has two advantages: it does not generate theside product from reaction at C2 of the epoxide (e.g., D, directlybelow), and avoids the generation of a mixture of stereoisomers. Todemonstrate that this strategy is feasible, we prepared a substrateanalogous to E and reacted this component with an amine, ultimatelygenerating the desired product (analogous to C).

Synthesis of (S)-C12-205 by Reductive Amination Approach

Step 1. Synthesis of Fragment 404 for the Reductive Amination Approach

(S)-2-((trityloxy)methyl)oxirane (401)

Trityl protected glycidol derivative 401 was prepared as describedpreviously (Schweizer, et al., Synthesis 2007, 3807-3814; which isincorporated herein by reference.) A solution of (R)-glycidol (5.0 g, 61mmol) in CH₂Cl₂ (30 mL) was added by syringe to a stirred solution oftrityl chloride (18.6 g, 66.8 mmol) and triethylamine (16.9 mL, 122mmol) in CH₂Cl₂ (67 mL) in an ice bath under argon. DMAP (742 mg, 6.08mmol) was added to the reaction mixture following addition of theglycidol. The reaction was allowed to warm to room temperature. After 14hours, the reaction mixture was diluted with 300 mL saturated aqueousNH₄Cl. The mixture was further diluted to ˜1 L with water to dissolveprecipitated salts. The product was extracted from the quenchingsolution with Et₂O (3×); combined ethereal layers were washed withbrine, dried over MgSO₄, filtered through paper, and concentrated byrotary evaporation to a white solid. The crude product was purified byrecrystallization from boiling MeOH (200 mL) affording the desiredproduct 401 (14.1 g, 73%) as white crystals. NMR analysis of thismaterial was consistent with that reported in the literature.(Schweizer, et al., Synthesis 2007, 3807-3814.)

(S)-2-(benzyloxy)dodecan-1-ol (403)

A 60 wt. % suspension of NaH in mineral oil (2.01 g, 50.3 mmol) wasadded to an oven-dried round bottom flask containing a magnetic stirbar. THF (120 mL) was added to the flask by syringe under Ar, and theflask was submerged in an ice bath. Crude 402 (14.9 g, 33.5 mmol) wasdissolved in anhydrous THF (50 mL) and was added slowly to the stirredsuspension of NaH. The reaction mixture was allowed to warm to roomtemperature. Benzyl chloride (5.8 mL, 50 mmol) was added to the reactionmixture. The flask was fitted with a reflux condenser, and the mixturewas warmed to reflux under Ar overnight. After the reaction mixture hadcooled, NH₄Cl (sat. aq., ˜300 mL) was added slowly to quench residualNaH. The suspension was transferred into a separatory funnel using H₂O(300 mL) and Et₂O (200 mL). The organic layer was extracted withadditional Et₂O; ethereal layers were dried over MgSO₄, filtered throughpaper, and concentrated to a yellow oil. This material was purified bychromatography on silica (gradient elution from hexanes to EtOAc);desired fractions were pooled and concentrated affording 15 g of aslightly yellow oil. This oil was dissolved in 1:1 MeOH/THF (100 mL).p-TsOH.H₂O (572 mg) was added to the mixture; the solution was stirredfor 6 hours. The reaction mixture was concentrated onto Celite by rotaryevaporation and purified by chromatography on silica gel (gradientelution from hexanes to ethyl acetate). Fractions containing the desiredproduct were pooled and concentrated affording 403 (5.44 g, 66%) as aclear oil. ¹H NMR (400 MHz, CDCl₃): δ 7.39-7.29 (m, 5H), 4.64 (d, J=11.6Hz, 1H), 4.55 (d, J=11.6 Hz, 1H), 3.74-3.67 (m, 1H), 3.60-3.49 (m, 2H),1.93-1.90 (m, 1H), 1.68-1.60 (m, 1H), 1.53-1.45 (m, 1H), 1.40-1.40 (m,16H), 0.89 (t, J=6.9 Hz, 3H).

(S)-2-(benzyloxy)dodecanal (404)

CH₂Cl₂ (10 mL) and oxalyl chloride (1.72 mL, 20.3 mmol) were added to anoven-dried 2-neck round bottom flask containing a magnetic stir barunder Ar. The flask was immersed in a dry ice/acetone bath. A solutionof DMSO (2.88 mL, 40.6 mmol) in CH₂Cl₂ (10 mL) was added to the stirredsolution of oxalyl chloride slowly. 403 (5.4 g, 18.5 mmol) was dissolvedin CH₂Cl₂ and added dropwise, over a period of 15 minutes, to the cold,stirred reaction mixture. After stirring for 2 hours, Et₃N (12.9 mL,18.46 mmol) was added to the reaction mixture, which was then allowed towarm to room temperature. The mixture was diluted with Et₂O (˜300 mL)and water. The ether layer was washed with sat. aq. NaHCO₃, 1M aq. HCl,and brine. The Et₂O layer was then dried over MgSO₄, filtered throughpaper, and concentrated by rotary evaporation. The crude product waspurified by chromatography on silica (gradient elution from hexanes to1:1 EtOAc/hexanes); fractions containing the desired product were pooledand concentrated affording 404 (3.58 g, 67%) as a clear, slightlyviscous oil. ¹H NMR (400 MHz, CDCl₃): δ 9.66 (d, J=1.9 Hz, 1H),7.37-7.31 (m, 5H), 4.69 (d, J=11.7 Hz, 1H), 4.55 (d, J=11.7 Hz, 1H),3.78-3.75 (m, 1H), 1.68 (dd, J=14.3, 7.2 Hz, 2H), 1.49-1.35 (m, 2H),1.25 (br s, 14H), 0.89 (t, J=6.7, 3H)

Step 2. Reductive Amination Pure Form of (S)-C12-205

1-(2-aminoethyl)piperazine (205, 39 μL, 0.3 mmol) was added to a vialcontaining a magnetic stir bar. MeOH (10 mL) and the aldehyde 404 (971mg, 3.34 mmol) were added to the vial. NaCNBH₃ (188 mg, 3 mmol) was thenadded to the mixture. Glacial AcOH was added dropwise to the stirredsolution until the pH (as measured using indicator strips) wasapproximately 5.5. The mixture bubbled during the addition of the AcOH.The mixture was stirred for 4 days, whereupon it was diluted with 1MNaOH (aq.) and CH₂Cl₂. The aqueous layer was extracted an additionaltime with CH₂Cl₂. The combined organic layers were washed with brine,dried over MgSO₄, filtered through paper and concentrated. The desiredintermediate was purified by chromatography on silica (CH₂Cl₂ to 10%MeOH/CH₂Cl₂) affording a yellow oil (83 mg). This oil was dissolved in25 mL 7:2:1 MeOH/H₂O/AcOH. A portion of 10 wt. % Pd/C was added to thesolution. The reaction mixture was stirred under H₂ (slightly aboveatmospheric pressure) for 8 hours. The reaction mixture was filteredthrough Celite to remove the Pd/C and then concentrated to a film. Massspectral analysis of this material indicated that it was the pure,desired product (S)-C12-205. MALDI-TOF-MS m/z: calcd for C₄₂H₈₈N₃O₃[M+H⁺], 682.7; found 682.9.

Having now described some illustrative embodiments of the invention, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the invention. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction. Use of ordinal terms such as “first”, “second”, “third”, etc.,in the claims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A method of preparing an aminoalcohol lipidoidcompound of the formula:

or a pharmaceutically acceptable salt, wherein: R_(A) and R_(F)independently are hydrogen,

R_(Y) and R_(Z) independently are hydrogen, each R₅ is independentlyselected from:

m is 1; and p is 1; the method comprising the step of reacting one ormore equivalents of an amine of the formula:

with one or more epoxide-containing compounds selected from:


2. The method of claim 1, wherein R_(A) is:


3. The method of claim 1, wherein R_(F) is:


4. The method of claim 1, wherein: RA is:

and R_(F) is:


5. The method of claim 1, wherein: RA is:

and R_(F) is:


6. The method of claim 1, wherein R_(Y) and R_(Z) independently arehydrogen, or


7. The method of claim 6, wherein R_(Y) and R_(Z) are


8. The method of claim 1, wherein R_(A) is:


9. The method of claim 1, wherein R_(F) is:


10. The method of claim 1, wherein the aminoalcohol lipidoid compound isselected from:

and pharmaceutically acceptable salts thereof.
 11. The method of claim10, wherein the aminoalcohol lipidoid compound is:

or a pharmaceutically acceptable salt thereof.
 12. The method of claim1, wherein the method comprises reacting one or more equivalents of anamine of the formula:

with an epoxide-containing compound of the formula:


13. The method of claim 12, wherein the aminoalcohol lipidoid compoundis:

or a pharmaceutically acceptable salt thereof.
 14. The method of claim12, wherein the epoxide-containing compound is:

and the aminoalcohol lipidoid compound is:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim1, wherein the step of reacting is performed with no solvent present.16. The method of claim 1, wherein the step of reacting is performed inthe presence of an aprotic solvent.
 17. The method of claim 1, whereinthe step of reacting is performed in the presence of a solvent selectedfrom tetrahydrofuran, diethyl ether, ethyl acetate, DMSO, DMF, methanol,ethanol, and water.
 18. The method of claim 1, wherein the step ofreacting is performed at a temperature ranging from 25° C. to 100° C.19. The method of claim 18, wherein the step of reacting is performed atapproximately 90° C.
 20. The method of claim 1, further comprising thestep of purifying the aminoalcohol lipidoid compound.